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Ghrelin variant protein |
| 7176292 |
Ghrelin variant protein
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| Patent Drawings: | |
| Inventor: |
Mintz |
| Date Issued: |
February 13, 2007 |
| Application: |
10/659,782 |
| Filed: |
September 11, 2003 |
| Inventors: |
Mintz; Liat (East Brunswick, NJ)
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| Assignee: |
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| Primary Examiner: |
Qian; Celine |
| Assistant Examiner: |
Dunston; Jennifer |
| Attorney Or Agent: |
Potter Anderson & Corroon LLPSafran; Jeffrey |
| U.S. Class: |
530/399; 536/23.51; 930/120; 930/20 |
| Field Of Search: |
530/350; 530/397; 530/399; 530/300 |
| International Class: |
A61K 38/22; C07K 14/00; C07H 21/04 |
| U.S Patent Documents: |
6291653; 6838438; 6967237 |
| Foreign Patent Documents: |
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| Other References: |
Gualillo et al. Ghrelin, a novel placental-derived hormone. Endocrinology. vol. 142, No. 2, pp. 788-794, Feb. 2001. cited by examiner.
Bednarek et al. Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J Med Chem. vol. 43, No. 23, pp. 4370-4376, Nov. 2000.cited by examiner.
Root et al. Clinical pharmacology of human growth hormone and its secretagogues. Curr Drug Targets Immune Endocr Metabol Disord. vol. 2, No. 1, pp. 27-52, Apr. 2002. cited by examiner.
Bednarek et al. Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J Med Chem. vol. 43, No. 23, pp. 4370-4376, Nov. 2000.cited by examiner.
Muccioli et al. Neuroendocrine and peripheral activities of ghrelin: implications in metabolism and obesity. Eur J Pharmacol. vol. 440, No. 2-3, pp. 235-254, Apr. 2002. cited by examiner.
Broglio et al. Natural and synthetic growth hormone secretagogues: do they have therapeutic protential? Treat Endocrinol. vol. 2, No. 3, pp. 153-163, 2003. cited by examiner.
A. Asakawa, et al., Antagonism of ghrelin receptor reduces food intake and body weight gain in mice, Gut 2003:52:947-952. cited by other.
Masayasu Kojima et al., Ghrelin is a growth-hormone-releasing acylated peptide from stomach, Nature, vol. 402, Dec. 9, 1999, pp. 656-660. cited by other.
Hiroshi Hosoda et al., Purification and Characterization of Rat des-Gin14-Ghrelin . . . , J. Biol. Chem., vol. 275, No. 29, Issue of Jul. 21, pp. 21995-22000, 2000. cited by other.
Mitsuyo Shintani et al., Ghrelin, and Endengenous Growth Hormone Secretagogue . . . , Diabetes, vol. 50, Feb. 2001, pp. 227-232. cited by other.
P L Jeffery et al., Expression and action of the growth hormone releasing peptide ghrelin . . . , Journal of Endocrinology (2002) 172, Feb 8, 2002, pp. R7-R11. cited by other.
Minoru Tanaka et al., Testis-specific and developmentally induced expression of a ghrelin . . . , Biochimica et Biophysica Acta, 1522 (2001) pp. 62-65. cited by other.
L. Trudel et al., Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus . . . , Am J Physiol Gastro Liver Physiol 282: G948-G952, 2002. cited by other.
Anke Hinney et al., Ghrelin gene: Identification of missense variants and a frameshift mutation . . . , J of Clinical Endocrinology & Metabolism 87(6): 2716-2719 (2002). cited by other.
Marta Korbonits et al., A variation in the ghrelin gene increases weight and decreases . . . , J of Clinical Endocrinology & Metabolism 87(8): 4005-4008 (2002). cited by other.
Olavi Ukkola et al., Role of Ghrelin Polymorphisms in Obesity Based on Three Different Studies, Obesity Research vol. 10 No. 8 Aug. 2002 pp. 782-791. cited by other.
Antonio Torsello et al., Short Ghrelin Peptides Neither Displace Ghrelin Binding In Vitro . . . , Endocrinology 143(5):1968-1971 (2002). cited by other.
David E. Cummings et al., A Preprandial Rise in Plasma Ghrelin Levels Suggests . . . , Diabetes, vol. 50, pp. 1714-1719, Aug. 2001. cited by other.
Kazuhiro Kawamura et al., Ghrelin Inhibits the Development of Mouse Preimplantation . . . , Endocrinology 144(6):2623-2633 (2003). cited by other.
A.M. Wren et al., The Novel Hypothalamic Peptide Ghrelin Stimulates Food Intake . . . , Endocrinology vol. 141, No. 11, pp. 4325-4328 (2000). cite- d by other. |
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| Abstract: |
The present invention concerns a protein produced from an alternative splice form of the human Ghrelin gene, an obesity and/or diabetes related gene. |
| Claim: |
The invention claimed is:
1. An isolated amino acid sequence coded by nucleotides 112 to 462 of SEQ ID NO:11.
2. The isolated amino acid sequence of claim 1, wherein said isolated amino acid sequence is SEQ ID NO:32. |
| Description: |
FIELD OF THE INVENTION
The present invention relates to obesity and diabetes markers, to reagents which can detect obesity and diabetes marker transcripts and translation products, to kits and methods for detecting obesity and diabetes marker transcripts andtranslation products, to methods and kits for screening and diagnosing obesity and diabetes in individuals and monitoring response to treatment, disease progression and disease recurrence in patients diagnosed with obesity and diabetes, to compoundswhich specifically bind to translation products of obesity and/or diabetes marker transcripts, to compositions for and methods of treating obesity and/or diabetes.
BACKGROUND OF THE INVENTION
Obesity is the second most important cause of preventable death in the United States, exceeded only by cigarette smoking. Obesity is estimated to affect 58,000,000 people and contribute to 300,000 deaths annually in the United States and itsprevalence is increasing. Individuals suffering from the disease are at increased risk of illness from hypertension, lipid disorders, coronary heart disease, type II diabetes, stroke, gall bladder disease, osteoarthritis, sleep apnea, respiratoryproblems and certain cancers.
Obesity develops when there is an excess of energy intake over energy usage. The causes of this excess may vary from patient to patient and are believed to stem from various genetic, social and environmental factors. Current research supportsthe view that under identical environmental conditions, different people gain weight at different rates and the amount they gain seems to be genetically determined. It has been proposed that natural selection caused our distant ancestors to acquire`thrifty genes` which boosted the ability to store fat from each feast in order to sustain the body through the next famine. In today's environment of a surfeit of high fat, high calorie `western style` food, `thrifty genes` have become a liability.
More and more scientists and physicians are coming to reject the traditional belief that poor diet and lack of exercise are solely to blame for obesity and are increasingly tending to view it as a medical condition. Health economists, usingprospective studies and national health statistics, have calculated the costs of obesity in the US in 1995 at $99.2 billion. By 2005 it is estimated that more than 120 million will be obese. The number of people living in France, Germany, Italy, the UKand the US could rise from 71 million in 1999 to 78 million in 2005. The economic impact of obesity in the US is now comparable to that of diabetes and ranks alongside expenditure on heart disease and hypertension. Medical researchers calculate that atleast 88% of all cases of type II diabetes, 57% of coronary heart disease cases, 11% of breast cancers, and 10% of colon cancers diagnosed in overweight Americans are attributable to obesity. The World health Organization has classified the obesitycondition as an epidemic, and has set up a special task force to tackle one of the greatest risks to human health and well-being. There remains a need for obesity and/or diabetes specific markers. There remains a need for reagents and kits which can beused to detect the presence of obesity and/or diabetes markers in samples from patients. There remains a need for methods of screening and diagnosing individuals who have obesity and/or diabetes and methods of monitoring response to treatment, diseaseprogression and disease recurrence in patients diagnosed with obesity and diabetes. There remains a need for reagents, kits and methods for determining the type of obesity and/or diabetes that an individual who is obese has. There remains a need forcompositions which can specifically target obesity and/or diabetes related cells. There remains a need for imaging agents which can specifically bind to obesity and/or diabetes cells. There remains a need for improved methods of imaging obesity and/ordiabetes cells. There remains a need for therapeutic agents which can specifically bind to obesity and/or diabetes cells. there remains a need for improved methods of treating individuals who are suspected of suffering from obesity and diabetes.
GLOSSARY
In the following description and claims use will be made, at times, with a variety of terms, and the meaning of such terms as they should be construed in accordance with the invention is as follows:
"Obesity and/or diabetes nucleic acid sequences"--the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 4 and of SEQ ID NO: 22 to SEQ ID NO: 25 sequences having at least 90% identity (see below) to said sequences and fragments (see below)of the above sequences of least 20 b.p. long. These sequences are sequences coding for naturally occurring, alternative splice variants of the native and known Adiponectin, depicted in Locus Link as locus Hs. 9370 under Accession NumberNM.sub.--004797 which is the sequence coding for the human 30 kDa glycoprotein of 244 amino acids. It should be emphasized that the novel variants of the present invention are naturally occurring sequences resulting from alternative splicing ofAdiponectin and not merely truncated, mutated or fragmented form of the gene.
the sequence shown in any one of SEQ ID NO: 5 to SEQ ID NO: 9 and of SEQ ID NO: 26 to SEQ ID NO: 30 sequences having at least 90% identity (see below) to said sequences and fragments (see below) of the above sequences of least 20 b.p. long. These sequences are sequences coding for naturally occurring, alternative splice variants of the native and known Adiponectin, depicted in Locus Link as locus Mm. 11450 under Accession Number NM.sub.--009605 which is the sequence coding for the mouse 30kDa glycoprotein of 247 amino acids. It should be emphasized that the novel variants of the present invention are naturally occurring sequences resulting from alternative splicing of Adiponectin and not merely truncated, mutated or fragmented form ofthe gene.
the sequence shown in any one of SEQ ID NO: 10 to SEQ ID NO: 11 and of SEQ ID NO: 31 to SEQ ID NO: 32 sequences having at least 90% identity (see below) to said sequences and fragments (see below) of the above sequences of least 20 b.p. long. These sequences are sequences coding for naturally occurring, alternative splice variants of the native and known Ghrelin, depicted in Locus Link as locus Hs. 51738 under Accession Number NM.sub.--016362 which is the sequence coding for the human 13 kDaglycoprotein of 117 amino acids. It should be emphasized that the novel variants of the present invention are naturally occurring sequences resulting from alternative splicing of Ghrelin and not merely truncated, mutated or fragmented form of the gene.
the sequence shown in any one of SEQ ID NO: 12 to SEQ ID NO: 18 and of SEQ ID NO: 33 to SEQ ID NO: 39 sequences having at least 90% identity (see below) to said sequences and fragments (see below) of the above sequences of least 20 b.p. long. These sequences are sequences coding for naturally occurring, alternative splice variants of the native and known 11-beta-HSD, depicted in Locus Link as locus Hs. 3290 under Accession Number NM.sub.--005525 which is the sequence coding for the human 32kDa glycoprotein of 292 amino acids. It should be emphasized that the novel variants of the present invention are naturally occurring sequences resulting from alternative splicing of 11-beta-HSD and not merely truncated, mutated or fragmented form ofthe gene.
the sequence shown in any one of SEQ ID NO: 19 to SEQ ID NO: 21 and of SEQ ID NO: 40 to SEQ ID NO: 42 sequences having at least 90% identity (see below) to said sequences and fragments (see below) of the above sequences of least 20 b.p. long. These sequences are sequences coding for naturally occurring, alternative splice variants of the native and known 11-beta-HSD, depicted in Locus Link as locus Mm. 15483 under Accession Number NM.sub.--008288 which is the sequence coding for the mouse 32kDa glycoprotein of 292 amino acids. It should be emphasized that the novel variants of the present invention are naturally occurring sequences resulting from alternative splicing of 11-beta-HSD and not merely truncated, mutated or fragmented form ofthe gene.
The description of the obesity and/or diabetes variants and their difference from the original sequence is summarized in Table 1 as follows:
TABLE-US-00001 TABLE 1 SEQ Obesity and GenBank GenBank ID Diabetes Human Mouse Gene NO: related genes Locus ID Locus ID Symbol Variation description 1 Adiponectin- 9370 11450 APM Nucleotide sequence of the WT (Variant human wild type protein 1)(human) 2 Adiponectin Nucleotide sequence of Variant 2 variant 2 (human) 3 Adiponectin Nucleotide sequence of Variant 3 variant 3 (human) 4 Adiponectin Nucleotide sequence of Variant 4 variant 4 (human) 5 Adiponectin- 9370 11450 APM Nucleotide sequenceof the WT (Variant mouse wild type protein 1) (mouse) 6 Adiponectin Nucleotide sequence of Variant 2 variant 2 (mouse) 7 Adiponectin Nucleotide sequence of Variant 3 variant 3 (mouse) 8 Adiponectin Nucleotide sequence of Variant 4 variant 4 (mouse) 9Adiponectin Nucleotide sequence of Variant 5 variant 5 (mouse) 10 Ghrelin- WT 51738 58991 GHRL Nucleotide sequence of the (variant 1) human wild type protein 11 Ghrelin Nucleotide sequence of Variant 2 variant 2 (human) 12 11-beta-HSD - 3290 15483HSD11B1 Nucleotide sequence of the WT wild type human protein (Variant 1) 13 11-beta-HSD Nucleotide sequence of Variant 2 variant 2 (human) 14 11-beta-HSD Nucleotide sequence of Variant 3 variant 3 (human) 15 11-beta-HSD Nucleotide sequence of Variant 4variant 4 (human) 16 11-beta-HSD Nucleotide sequence of Variant 5 variant 5 (human) 17 11-beta-HSD Nucleotide sequence of Variant 6 variant 6 (human) 18 11-beta-HSD Nucleotide sequence of Variant 7 variant 7 (human) 19 11-beta-HSD - Nucleotide sequenceof the WT mouse wild type protein (Variant 1) 20 11-beta-HSD Nucleotide sequence of Variant 8 variant 8 (mouse) 21 11-beta-HSD Nucleotide sequence of Variant 9 variant 9 (mouse) 22 Adiponectin- 9370 11450 APM Wild type human protein WT (Variant sequence1) 23 Adiponectin Alternative initiation (human) Variant 2 24 Adiponectin Variant 3 25 Adiponectin Variant 4 26 Adiponectin- 9370 11450 APM Wild type mouse protein WT (Variant sequence 1) 27 Adiponectin Alternative initiation (mouse) Variant 2 28Adiponectin Alternative 45 amino acids Variant 3 from position 111 in the wild type protein creating a variant with 156 amino acids (mouse) 29 Adiponectin Alternative 58 amino acids Variant 4 from position 111 in the wild type protein creating a variantwith 169 amino acids (mouse) 30 Adiponectin Truncated variant 76 amino Variant 5 acids long (mouse) 31 Ghrelin- WT 51738 58991 GHRL Wild type human protein (variant 1) sequence 32 Ghrelin Alternative 70 amino acids Variant 2 from position 35 in the wildtype protein creating a variant with 117 amino acids (human) 33 11-beta-HSD - 3290 15483 HSD11B1 Wild type human protein WT sequence (Variant 1) 34 11-beta-HSD Deletion of 18 amino acids Variant 2 from amino acid 64 in the wild type protein and analternative exon of 16 amino acids replacing the rest of the amino acids from amino acid 165 in the wild type protein (human) 35 11-beta-HSD Alternative 9 amino acids Variant 3 from amino acid 286 creating a variant with 295 amino acids (human) 3611-beta-HSD Deletion of 18 amino acids Variant 4 from amino acid 137 till amino acid 155 in the wild type protein (human) 37 11-beta-HSD Deletion of 20 amino acids Variant 5 from amino acid 64 till amino acid 84 in the wild type protein (human) 3811-beta-HSD Alternative initiation at amino Variant 6 acid no. 31 in the wild type protein (human) 39 11-beta-HSD Deletion of 48 amino acids Variant 7 from amino acid 173 till amino acid 221 in the wild type protein (human) 40 11-beta-HSD - Wild typemouse protein WT sequence (Variant 1) 41 11-beta-HSD Deletion of 32 amino acids Variant 8 from amino acid 29 till amino acid 71 in the wild type protein 42 11-beta-HSD Alternative 19 amino acids Variant 9 from amino acid 173 creating a variant with 192amino acids (mouse) SEQ ID NOS: 1 21 are nucleotide sequences. SEQ ID NOS: 22 42 are protein sequences encoded by SEQ ID NOS 1 21.
TABLE-US-00002 TABLE 2 SEQ ID 1 9 Adiponectin variants: SEQ ID NO. 1: NM_004797_T1 | Length 4517 CTGATTCCATACCAGAGGGGCTCAGGATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAG CTCTGCCCGGGCATGACCAGGAAACCACGACTCAAGGGCCCGGAGTCCTGCTTCCCCTGCCCAAGGGGGCCTGCACAGGTTGGATGGCGGGCATCCCAGGGCATCCGGGCCATAATGGGG CCCCAGGCCGTGATGGCAGAGATGGCACCCCTGGTGAGAAGGGTGAGAAAGGAGATCCAG GTCTTATTGGTCCTAAGGGAGACATCGGTGAAACCGGAGTACCCGGGGCTGAAGGTCCCC GAGGCTTTCCGGGAATCCAAGGCAGGAAAGGAGAACCTGGAGAAGGTGCCTATGTATACCGCTCAGCATTCAGTGTGGGATTGGAGACTTACGTTACTATCCCCAACATGCCCATTCGCT TTACCAAGATCTTCTACAATCAGCAAAACCACTATGATGGCTCCACTGGTAAATTCCACT GCAACATTCCTGGGCTGTACTACTTTGCCTACCACATCACAGTCTATATGAAGGATGTGA AGGTCAGCCTCTTCAAGAAGGACAAGGCTATGCTCTTCACCTATGATCAGTACCAGGAAAATAATGTGGACCAGGCCTCCGGCTCTGTGCTCCTGCATCTGGAGGTGGGCGACCAAGTCT GGCTCCAGGTGTATGGGGAAGGAGAGCGTAATGGACTCTATGCTGATAATGACAATGACT CCACCTTCACAGGCTTTCTTCTCTACCATGACACCAACTGATCACCACTAACTCAGAGCC TCCTCCAGGCCAAACAGCCCCAAAGTCAATTAAAGGCTTTCAGTACGGTTAGGAAGTTGATTATTATTTAGTTGGAGGCCTTTAGATATTATTCATTCATTTACTCATTCATTTATTCAT TCATTCATCAAGTAACTTTAAAAAAATCATATGCTATGTTCCCAGTCCTGGGGAGCTTCA CAAACATGACCAGATAACTGACTAGAAAGAAGTAGTTGACAGTGCTATTTTGTGCCCACT GTCTCTCCTGATGCTCATATCAATCCTATAAGGCACAGGGAACAAGCATTCTCCTGTTTTTACAGATTGTATCCTGAGGCTGAGAGAGTTAAGTGAATGTCTAAGGTCACACAGTATTAA GTGACAGTGCTAGAAATCAAACCCAGAGCTGTGGACTTTGTTCACTAGACTGTGCCCTTT TATAGAGGTACATGTTCTCTTTGGAGTGTTGGTAGGTGTCTGTTTCCCACCTCACCTGAG AGCCATTGAATTTGCCTTCCTCATGAATTAAAACCTCCCCCAAGCAGAGCTTCCTCAGAGAAAGTGGTTCTATGATGAAGTCCTGTCTTGGAAGGACTACTACTCAATGGCCCCTGCACT ACTCTACTTCCTCTTACCTATGTCCCTTCTCATGCCTTTCCCTCCAACGGGGAAAGCCAA CTCCATCTCTAAGTGCTGAACTCATCCCTGTTCCTCAAGGCCACCTGGCCAGGAGCTTCT CTGATGTGATATCCACTTTTTTTTTTTTTTGAGATGGAGTCTCACTCTGTCACCCAGGCTGGAGTACAGTGACACGACCTCGGCTCACTGCAGCCTCCTTCTCCTGGGTCCAAGCAATTA TTGTGCCTCAGCCTCCCGAGTAGCTGAGACTTCAGGTGCATTCCACCACACATGGCTAAT TTTTGTATTTTTAGTAGAAATGGGGTTTCGTCATGTTGGCCAGGCTGGTCTCGAACTCCT GGCCTAGGTGATCCACCCGCCTCGACCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCATGCCCAGTCGATATCTCACTTTTTATTTTGCCATGGATGAGAGTCCTGGGTGTGAGGA ACACCTCCCACCAGGCTAGAGGCAACTGCCCACGAAGGACTGTGCTTCCGTCACCTCTAA ATCCCTTGCAGATCCTTGATAAATGCCTCATGAAGACCAATCTCTTGAATCCCATATCTA CCCAGAATTAACTCCATTCCAGTCTCTGCATGTAATCAGTTTTATCCACAGAAACATTTTCATTTTAGGAAATCCCTGGTTTAAGTATCAATCCTTGTTCAGCTGGACAATATGAATCTT TTCCACTGAAGTTAGGGATGACTGTGATTTTCAGAACACGTCCAGAATTTTTCATCAAGA AGGTAGCTTGAGCCTGAAATGCAAAACCCATGGAGGAATTCTGAAGCCATTGTCTCCTTG AGTACGAACAGGGTCAGGGAAGACTGGGCCTCCTGAATTTATTATTGTTCTTTAAGAATTACAGGTTGAGGTAGTTGATGGTGGTAAACATTCTCTCAGGAGACAATAACTCCAGTGATG TTTTTCAAAGATTTTAGCAAAAACAGAGTAAATAGCATTCTCTATCAATATATAAATTTA AAAAACTATCTTTTTGCTTACAGTTTTAAATTCTGAACAATTTCTCTTATATGTGTATTG CTAATCATTAAGGTATTATTTTTTCCACATATAAAGCTTTGTCTTTTTGTTGTTGTTGTTGTTTTTAAGATGGAGTTTCCCTCTGTTCCCAGGCTAGAGTGCAGTGGCATGATCTCGGCT TACTGCAACCTTTGCCTCCCACGTTTAAGCGATTCTTCTGCCTCAGCCTCCCGAGTAGCT GGGACCACACGTGCCTACCACCATGCCAGGCTAATTTTTGTATTTTTAGTAAAGACAGGG TTTCACCATATTGGCCAGGCTGGTCTCGAACTCCTGACCTTGTGATCTGCCCGCCTCCATTGTGTTGTTATTTGTGAGAAAGATAGATATGAGGTTTAGAGAGGGATGAAGAGGTGAGAG TAAGCCTTGTGTTAGTCAGAACTCTGTGTTGTGAATGTCATTCACAACAGAAAACCCAAA ATATTATGCAAACTACTGTAAGCAAGAAAAATAAAGGAAAAATGGAAACATTTATTCCTT TGCATAATAGAAATTACCAGAGTTGTTCTGTCTTTAGATAAGGTTTGAACCAAAGCTCAAAACAATCAAGACCCTTTTCTGTATGTCCTTCTGTTCTGCCTTCCGCAGTGTAGGCTTTAC CCTCAGGTGCTACACAGTATAGTTCTAGGGTTTCCCTCCCGATATCAAAAAGACTGTGGC CTGCCCAGCTCTCGTATCCCCAAGCCACACCATCTGGCTAAATGGACATCATGTTTTCTG GTGATGCCCAAAGAGGAGAGAGGAAGCTCTCTTTCCCAGATGCCCCAGCAAGTGTAACCTTGCATCTCATTGCTCTGGCTGAGTTGTGTGCCTGTTTCTGACCAATCACTGAGTCAGGAG GATGAAATATTCATATTGACTTAATTGCAGCTTAAGTTAGGGGTATGTAGAGGTATTTTC CCTAAAGCAAAATTGGGACACTGTTATCAGAAATAGGAGAGTGGATGATAGATGCAAAAT AATACCTGTCCACAACAAACTCTTAATGCTGTGTTTGAGCTTTCATGAGTTTCCCAGAGAGACATAGCTGGAAAATTCCTATTGATTTTCTCTAAAATTTCAACAAGTAGCTAAAGTCTG GCTATGCTCACAGTCTCACATCTGGTGGGGGTGGGCTCCTTACAGAACACGCTTTCACAG TTACCCTAAACTCTCTGGGGCAGGGTTATTCCTTTGTGGAACCAGAGGCACAGAGACAGT CAACTGAGGCCCAACAGAGGCCTGAGAGAAACTGAGGTCAAGATTTCAGGATTAATGGTCCTGTGATGCTTTGAAGTACAATTGTGGATTTGTCCAATTCTCTTTAGTTCTGTCAGCTTT TGCTTCATATATTTTAGCGCTCTATTATTAGATATATACATGTTTAGTATTATGTCTTAT TGGTGCATTTACTCTCTTATCATTATGTAATGTCCTTCTTTATCTGTGATAATTTTCTGT GTTCTGAAGTCTACTTTGTCTAAAAATAACATACGCACTCAACTTCCTTTTCTTTCTTCCTTCCTTTCTTTCTTCCTTCCTTTCTTTCTCTCTCTCTCTTTCCTTCCTTCCTTCCTCCTT TTCTCTCTCTCTCTCTCTCTCTCTCTTTTCTTGACAGACTCTCGTTCTGTGGCCCTGGCT GGAGTTCAGTGGTGTGATCTTGGCTCACTGCTACCTCTACCATGAGCAATTCTCCTGCCT CAGCCTCCCAAGTAGCTGGAACTACAGGCTCATGCCACTGCGCCCAGCTAATTTTTGTATTTTTCGTAGAGACGGGGTTTCACCACATTCGTCAGGTTGGTTTCAAACTCCTGACTTTGT GATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCATCACACCTGG TCAACTTTCTTTTGATTAGTGTTTTTGTGGTATATCTTTTTCCATCATGTTACTTTAAAT ATATCTATATTATTGTATTTAAAATGTGTTTCTTACAGAGTGCATGTAGTTGGGTATAATTTTTATCCAGTCTAAAAATATCTGTCTTTTAATTGGTGTTTAGACAATTTATATTTAATA AAATGGTGGAATTTAAA SEQ ID NO. 2: NM_004797_T2 | Length 484 ATGACCCGGGGCTGAAGGTCCCCGAGGCTTTCCGGGAATCCAAGGCAGGAAAGGAGAACC TGGAGAAGGTGCCTATGTATACCGCTCAGCATTCAGTGTGGGATTGGAGACTTACGTTACTATCCCCAACATGCCCATTCGCTTTACCAAGATCTTCTACAATCAGCAAAACCACTATGA TGGCTCCACTGGTAAATTCCACTGCAACATTCCTGGGCTGTACTACTTTGCCTACCACAT CACAGTCTATATGAAGGATGTGAAGGTCAGCCTCTTCAAGAAGGACAAGGCTATGCTCTT CACCTATGATCAGTACCAGGAAAATAATGTGGACCAGGCCTCCGGCTCTGTGCTCCTGCATCTGGAGGTGGGCGACCAAGTCTGGCTCCAGGTGTATGGGGAAGGAGAGCGTAATGGACT CTATGCTGATAATGACAATGACTCCACCTTCACAGGCTTTCTTCTCTACCATGACACCAA CTGA SEQ ID NO. 3: NM_004797_T3 | Length 718 CTGATTCCATACCAGAGGGGCTCAGGATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAGCTCTGCCCGGGCATGACCAGGAAACCACGACTCAAGGGCCCGGAGTCCTGCTTCCCCTGC CCAAGGGGGCCTGCACAGGTTGGATGGCGGGCATCCCAGGGCATCCGGGCCATAATGGGG CCCCAGGCCGTGATGGCAGAGATGGCACCCCTGGTGAGAAGGGTGAGAAAGGAGATCCAG GTCTTATTGGTCCTAAGGGAGACATCGGTGAAACCGGAGTACCCGGGGCTGAAGGTCCCCGAGGCTTTCCGGGAATCCAAGGCAGGAAAGGAGAACCTGGAGAAGGTGCGTTACTATCCC CAACATGCCCATTCGCTTTACCAAGATCTTCTACAATCAGCAAAACCACTATGATGGCTC CACTGGTAAATTCCACTGCAACATTCCTGGGCTGTACTACTTTGCCTACCACATCACAGT CTATATGAAGGATGTGAAGGTCAGCCTCTTCAAGAAGGACAAGGCTATGCTCTTCACCTATGATCAGTACCAGGAAAATAATGTGGACCAGGCCTCCGGCTCTGTGCTCCTGCATCTGGA GGTGGGCGACCAAGTCTGGCTCCAGGTGTATGGGGAAGGAGAGCGTAATGGACTCTATGC TGATAATGACAATGACTCCACCTTCACAGGCTTTCTTCTCTACCATGACACCAACTGA SEQ ID NO. 4: NM_004797_T4 | Length 537CTGATTCCATACCAGGGGGGCTCAGGATGCTGTTGCTGGGAGCTGTTCTACTGCTATTAG CTCTGCCCGGGCATGACCAGGAAACCACGACTCAAGGGCCCGGAGTCCTGCTTCCCCTGC CCAAGGGGGCCTGCACAGGTTGGATGGCGGGCATCCCAGGGCATCCGGGCCATAATGGGG CCCCAGGCCGTGATGGCAGAGATGGCACCCCTGGTGAGAAGGGTGAGAAAGGAGATCCAGGTCTTATTGGTCCTAAGGGAGACATCGGTGAAACCGGAGTACCCGGGGCTGAAGGTCCCC GAGGCTTTCCGGGAATCCAAGGCAGGAAAGGAGAACCTGGAGAAGGTGCCTATGTATACC GCTCAGCATTCAGTGTGGGATTGGAGACTTACGTTACTATCCCCAACATGCCCATTCGCT TTACCAAGATCTTCTACAATCAGCAAAACCACTATGATGGCTCCACTGGTAAATTCCACTGCAACATTCCTGGGCTGTACCTTCACAGGCTTTCTTCTCTACCATGACACCAACTGA SEQ ID NO. 5: U37222_T1 | Length: 1306 WT ATGAGACCTGGCCACTTTCTCCTCATTTCTGTCTGTACGATTGTCAGTGGATCTGACGAC ACCAAAAGGGCTCAGGATGCTACTGTTGCAAGCTCTCCTGTTCCTCTTAATCCTGCCCAGTCATGCCGAAGATGACGTTACTACAACTGAAGAGCTAGCTCCTGCTTTGGTCCCTCCACC CAAGGGAACTTGTGCAGGTTGGATGGCAGGCATCCCAGGACATCCTGGCCACAATGGCAC ACCAGGCCGTGATGGCAGAGATGGCACTCCTGGAGAGAAGGGAGAGAAAGGAGATGCAGG TCTTCTTGGTCCTAAGGGTGAGACAGGAGATGTTGGAATGACAGGAGCTGAAGGGCCACGGGGCTTCCCCGGAACCCCTGGCAGGAAAGGAGAGCCTGGAGAAGCCGCTTATGTGTATCG CTCAGCGTTCAGTGTGGGGCTGGAGACCCGCGTCACTGTTCCCAATGTACCCATTCGCTT TACTAAGATCTTCTACAACCAACAGAATCATTATGACGGCAGCACTGGCAAGTTCTACTG CAACATTCCGGGACTCTACTACTTCTCTTACCACATCACGGTGTACATGAAAGATGTGAAGGTGAGCCTCTTCAAGAAGGACAAGGCCGTTCTCTTCACCTACGACCAGTATCAGGAAAA GAATGTGGACCAGGCCTCTGGCTCTGTGCTCCTCCATCTGGAGGTGGGAGACCAAGTCTG GCTCCAGGTGTATGGGGATGGGGACCACAATGGACTCTATGCAGATAACGTCAACGACTC TACATTTACTGGCTTTCTTCTCTACCATGATACCAACTGACTGCAACTACCCATAGCCCATACACCAGGAGAATCATGGAACAGTCGACACACTTTCAGCTTAGTTTGAGAGATTGATTT TATTGCTTAGTTTGAGAGTCCTGAGTATTATCCACACGTGTACTCACTTGTTCATTAAAC
GACTTTATAAAAAATAATTTGTGTTCCTAGTCCAGAAAAAAAGGCACTCCCTGGTCTCCA CGACTCTTACATGGTAGCAATAACAGAATGAAAATCACATTTGGTATGGGGGCTTCACAA TATTCGCATGACTGTCTGGAAGTAGACCATGCTATTTTTCTGCTCACTGTACACAAATAT TGTTCACATAAACCCTATAATGTAAATATGAAATACAGTGATTACTCTTCTCACAGGCTGASTGTATGAATTCTAAAGACCCATAAGTATTAAAGTGGTAGGGATAAATTGGAAAAAAAA AAAAAAAAAAAGAAAAACTTTAGAGCACACTGGCGGCCGTTACTAG SEQ ID NO. 6: U37222_T2 | LENGTH: 1184 GCTCATTCATCTTTTAATTCACCCATAAAGGCTTTGAAAACTAAGGCTGGAGATGAACTTATAGGAGCCTGCCAGGCCGTGGAGAGTGAGGAAGCAGAGATGACGGAGATGATGTCTTTC CTTGTCCTGTGAAATGGATTGTGGGTAGAGGTTCCGGAGATAATGCCTCTTGCTGGAAAC AGTCTGGGCAGTTCTGTTCCCGCCATTCACAGAATTCTTCTCACTTTCTAGGTCTTCTTG GTCCTAAGGGTGAGACAGGAGATGTTGGAATGACAGGAGCTGAAGGGCCACGGGGCTTCCCCGGAACCCCTGGCAGGAAAGGAGAGCCTGGAGAAGCCGCTTATGTGTATCGCTCAGCGT TCAGTGTGGGGCTGGAGACCCGCGTCACTGTTCCCAATGTACCCATTCGCTTTACTAAGA TCTTCTACAACCAACAGAATCATTATGACGGCAGCACTGGCAAGTTCTACTGCAACATTC CGGGACTCTACTACTTCTCTTACCACATCACGGTGTACATGAAAGATGTGAAGGTGAGCCTCTTCAAGAAGGACAAGGCCGTTCTCTTCACCTACGACCAGTATCAGGAAAAGAATGTGG ACCAGGCCTCTGGCTCTGTGCTCCTCCATCTGGAGGTGGGAGACCAAGTCTGGCTCCAGG TGTATGGGGATGGGGACCACAATGGACTCTATGCAGATAACGTCAACGACTCTACATTTA CTGGCTTTCTTCTCTACCATGATACCAACTGACTGCAACTACCCATAGCCCATACACCAGGAGAATCATGGAACAGTCGACACACTTTCAGCTTAGTTTGAGAGATTGATTTTATTGCTT AGTTTGAGAGTCCTGAGTATTATCCACACGTGTACTCACTTGTTCATTAAACGACTTTAT AAAAAATAATTTGTGTTCCTAGTCCAGAAAAAAAGGCACTCCCTGGTCTCCACGACTCTT ACATGGTAGCAATAACAGAATGAAAATCACATTTGGTATGGGGGCTTCACAATATTCGCATGACTGTCTGGAAGTAGACCATGCTATTTTTCTGCTCACTGTACACAAATATTGTTCACA TAAACCCTATAATGTAAATATGAAATACAGTGATTACTCTTCTCACAGGCTGAGTGTATG AATTCTAAAGACCCATAAGTATTAAAGTGGTAGGGATAAATTGG SEQ ID NO. 7: U37222_T3 | LENGTH: 1209ATGAGACCTGGCCACTTTCTCCTCATTTCTGTCTGTACGATTGTCAGTGGATCTGACGAC ACCAAAAGGGCTCAGGATGCTACTGTTGCAAGCTCTCCTGTTCCTCTTAATCCTGCCCAG TCATGCCGAAGATGACGTTACTACAACTGAAGAGCTAGCTCCTGCTTTGGTCCCTCCACC CAAGGGAACTTGTGCAGGTTGGATGGCAGGCATCCCAGGACATCCTGGCCACAATGGCACACCAGGCCGTGATGGCAGAGATGGCACTCCTGGAGAGAAGGGAGAGAAAGGAGATGCAGG TCTTCTTGGTCCTAAGGGTGAGACAGGAGATGTTGGAATGACAGGAGCTGAAGGGCCACG GGGCTTCCCCGGAACCCCTGGCAGGAAAGGAGAGCCTGGAGAAGCCGCGTCACTGTTCCC AATGTACCCATTCGCTTTACTAAGATCTTCTACAACCAACAGAATCATTATGACGGCAGCACTGGCAAGTTCTACTGCAACATTCCGGGACTCTACTACTTCTCTTACCACATCACGGTG TACATGAAAGATGTGAAGGTGAGCCTCTTCAAGAAGGACAAGGCCGTTCTCTTCACCTAC GACCAGTATCAGGAAAAGAATGTGGACCAGGCCTCTGGCTCTGTGCTCCTCCATCTGGAG GTGGGAGACCAAGTCTGGCTCCAGGTGTATGGGGATGGGGACCACAATGGACTCTATGCAGATAACGTCAACGACTCTACATTTACTGGCTTTCTTCTCTACCATGATACCAACTGACTG CAACTACCCATAGCCCATACACCAGGAGAATCATGGAACAGTCGACACACTTTCAGCTTA GTTTGAGAGATTGATTTTATTGCTTAGTTTGAGAGTCCTGAGTATTATCCACACGTGTAC TCACTTGTTCATTAAACGACTTTATAAAAAATAATTTGTGTTCCTAGTCCAGAAAAAAAGGCACTCCCTGGTCTCCACGACTCTTACATGGTAGCAATAACAGAATGAAAATCACATTTG GTATGGGGGCTTCACAATATTCGCATGACTGTCTGGAAGTAGACCATGCTATTTTTCTGC TCACTGTACACAAATATTGTTCACATAAACCCTATAATGTAAATATGAAATACAGTGATT ACTCTTCTCACAGGCTGAGTGTATGAATTCTAAAGACCCATAAGTATTAAAGTGGTAGGG ATAAATTGGSEQ ID NO. 8: U37222_T4 | LENGTH: 1028 ATGAGACCTGGCCACTTTCTCCTCATTTCTGTCTGTACGATTGTCAGTGGATCTGACGAC ACCAAAAGGGCTCAGGATGCTACTGTTGCAAGCTCTCCTGTTCCTCTTAATCCTGCCCAG TCATGCCGAAGATGACGTTACTACAACTGAAGAGCTAGCTCCTGCTTTGGTCCCTCCACCCAAGGGAACTTGTGCAGGTTGGATGGCAGGCATCCCAGGACATCCTGGCCACAATGGCAC ACCAGGCCGTGATGGCAGAGATGGCACTCCTGGAGAGAAGGGAGAGAAAGGAGATGCAGG TCTTCTTGGTCCTAAGGGTGAGACAGGAGATGTTGGAATGACAGGAGCTGAAGGGCCACG CGGCTTCCCCGGAACCCCTGGCAGGAAAGGAGAGCCTGGAGAAGCCGCTTATGTGTATCGCTCAGCGTTCAGTGTGGGGCTGGAGACCCGCGTCACTGTTCCCAATGTACCCATTCGCTT TACTAAGATCTTCTACAACCAACAGAATCATTATGACGGCAGCACTGGCAAGTTCTACTG CAACATTCCGGGACTCTACATTTACTGGCTTTCTTCTCTACCATGATACCAACTGACTGC AACTACCCATAGCCCATACACCAGGAGAATCATGGAACAGTCGACACACTTTCAGCTTAGTTTGAGAGATTGATTTTATTGCTTAGTTTGAGAGTCCTGAGTATTATCCACACGTGTACT CACTTGTTCATTAAACGACTTTATAAAAAATAATTTGTGTTCCTAGTCCAGAAAAAAAGG CACTCCCTGGTCTCCACGACTCTTACATGGTAGCAATAACAGAATGAAAATCACATTTGG TATGGGGGCTTCACAATATTCGCATGACTGTCTGGAAGTAGACCATGCTATTTTTCTGCTCACTGTACACAAATATTGTTCACATAAACCCTATAATGTAAATATGAAATACAGTGATTA CTCTTCTCACAGGCTGAGTGTATGAATTCTAAAGACCCATAAGTATTAAAGTGGTAGGGA TAAATTGG SEQ ID NO. 9: U37222_T5 | LENGTH: 306 ATGAGACCTGGCCACTTTCTCCTCATTTCTGTCTGTACGATTGTCAGTGGATCTGACGACACCAAAAGGGCTCAGGATGCTACTGTTGCAAGCTCTCCTGTTCCTCTTAATCCTGCCCAG TCATGCCGAAGATGACGTTACTACAACTGAAGAGCTAGCTCCTGCTTTGGTCCCTCCACC CAAGGGAACTTGTGCAGGTTGGATGGCAGGCATCCCAGGACATCCTGGCCACATAAAAAT ATAATTCGAGGGGCATCCACCAGGCCGGCTGAATTGTGCCAAAATATGGCACTTCCTGCA AGATAA SEQID 10 11 Ghrelin variants: SEQ ID NO. 10: NM_016362_T1 | Length: 665 ACTCTGGATGGGTGCTGTTTAGACAAACGCCGTCTCCTATATAAGACCTGACAGCACAGG CACCACTCCGCCAGGACTGCAGGCCCACCTGTCTGCAACCCAGCTGAGGCCATGCCCTCC CCAGGGACCGTCTGCAGCCTCCTGCTCCTCGGCATGCTCTGGCTGGACTTGGCCATGGCAGGCTCCAGCTTCCTGAGCCCTGAACACCAGAGAGTCCAGCAGAGAAAGGAGTCGAAGAAG CCACCAGCCAAGCTGCAGCCCCGAGCTCTAGCAGGCTGGCTCCGCCCGGAAGATGGAGGT CAAGCAGAAGGGGCAGAGGATGAACTGGAAGTCCGGTTCAACGCCCCCTTTGATGTTGGA ATCAAGCTGTCAGGGGTTCAGTACCAGCAGCACAGCCAGGCCCTGGGGAAGTTTCTTCAGGACATCCTCTGGGAAGAGGCCAAAGAGGCCCCAGCCGACAAGTGATCGCCCACAAGCCTT ACTCACCTCTCTCTAAGTTTAGAAGCGCTCATCTGGCTTTTCGCTTGCTTCTGCAGCAAC TCCCACGACTGTTGTACAAGCTCAGGAGGCGAATAAATGTTCAAACTGTATGCTGATGTT CCAAATGGGAATTTATTTCAAAGAGGAAAAGTTAATATTTTACTTTAAAAAAATCAAAAT AATAC SEQID NO. 11: NM_016362_T2 | Length: 579 ACTCTGGATGGGTGCTGTTTAGACAAACGCCGTCTCCTATATAAGACCTGACAGCACAGG CACCACTCCGCCAGGACTGCAGGCCCACCTGTCTGCAACCCAGCTGAGGCCATGCCCTCC CCAGGGACCGTCTGCAGCCTCCTGCTCCTCGGCATGCTCTGGCTGGACTTGGCCATGGCAGGCTCCAGCTTCCTGAGCCCTGAACACCAGAGAGTCCAGGTGAGACCTCCCCACAAAGCC CCACATGTTGTTCCAGCCCTGCCACTTAGCAACCAGCTCTGTGACCTGGAGCAGCAGCGC CATCTCTGGGCTTCAGTCTTCTCCCAGAGCACAAAGGACTCTGGGTCTGACCTCACTGTT TCTGGAAGGACATGGGGGCTTAGAGTCCTAAACAGACTGTTTCCCCCTTCCAGCAGAGAAAGGAGTCGAAGAAGCCACCAGCCAAGCTGCAGCCCCGAGCTCTAGCAGGCTGGCTCCGCC CGGAAGATGGAGGTCAAGCAGAAGGGGCAGAGGATGAACTGGAAGTCCGGGTCGGTACCT CTGCAGTTTTATGCTTCTGTGGCAGCGAGGAGGGTGGGG SEQ ID 12 21 HSD11B variants: SEQ ID 12: NM_005525_T1 WT| Length: 1448GCACTGCCTGAGACTACTCCAGCCTCCCCCGTCCCTGATGTCACAATTCAGAGGCTGCTG CCTGCTTAGGAGGTTGTAGAAAGCTCTGTAGGTTCTCTCTGTGTGTCCTACAGGAGTCTT CAGGCCAGCTCCCTGTCGGATGGCTTTTATGAAAAAATATCTCCTCCCCATTCTGGGGCT CTTCATGGCCTACTACTACTATTCTGCAAACGAGGAATTCAGACCAGAGATGCTCCAAGGAAAGAAAGTGATTGTCACAGGGGCCAGCAAAGGGATCGGAAGAGAGATGGCTTATCATCT GGCGAAGATGGGAGCCCATGTGGTGGTGACAGCGAGGTCAAAAGAAACTCTACAGAAGGT GGTATCCCACTGCCTGGAGCTTGGAGCAGCCTCAGCACACTACATTGCTGGCACCATGGA AGACATGACCTTCGCAGAGCAATTTGTTGCCCAAGCAGGAAAGCTCATGGGAGGACTAGACATGCTCATTCTCAACCACATCACCAACACTTCTTTGAATCTTTTTCATGATGATATTCA CCATGTGCGCAAAAGCATGGAAGTCAACTTCCTCAGTTACGTGGTCCTGACTGTAGCTGC CTTGCCCATGCTGAAGCAGAGCAATGGAAGCATTGTTGTCGTCTCCTCTCTGGCTGGGAA AGTGGCTTATCCAATGGTTGCTGCCTATTCTGCAAGCAAGTTTGCTTTGGATCGGTTCTTCTCCTCCATCAGAAAGGAATATTCAGTGTCCAGGGTCAATGTATCAATCACTCTCTGTGT TCTTGGCCTCATAGACACAGAAACAGCCATGAAGGCAGTTTCTCGGATAGTCCATATGCA AGCAGCTCCAAAGGAGGAATGTGCCCTGGAGATCATCAAAGGGGGAGCTCTGCGCCAAGA AGAAGTGTATTATGACAGCTCACTCTGGACCACTCTTCTGATCAGAAATCCATGCAGGAAGATCCTGGAATTTCTCTACTCAACGAGCTATAATATGGACAGATTCATAAACAAGTAGGA ACTCCCTGAGGGCTGGGCATGCTGAGGGATTTTGGGACTGTTCTGTCTCATGTTTATCTG AGCTCTTATCTATGAAGACATCTTCCCAGAGTGTCCCCAGAGACATGCAAGTCATGGGTC ACACCTGACAAATGGAAGGAGTTCCTCTAACATTTGCAAAATGGAAATGTAATAATAATGAATGTCATGCACCGCTGCAGCCAGCAGTTGTAAAATTGTTAGTAAACATAGGTATAATTA CCAGATAGTTATATTAAATTTATATCTTATATATAATAATATGTGATGATTAATACAATA TTAATTATAATAAAGGTCACATAAACTTTATAAATTCATAACTGGTAGCTATAACTTGAG CTTATTCAGGATGGTTTCTTTAAAACCATAAACTGTACAAATGAAATTTTTCAATATTTG TTTCTTATSEQ ID 13: NM_005525_T2 | LENGTH: 708 GCACTGCCTGAGACTACTCCAGCCTCCCCCGTCCCTGATGTCACAATTCAGAGGCTGCTG CCTGCTTAGGAGGTTGTAGAAAGCTCTGTAGGTTCTCTCTGTGTGTCCTACAGGAGTCTT
CAGGCCAGCTCCCTGTCGGATGGCTTTTATGAAAAAATATCTCCTCCCCATTCTGGGGCT CTTCATGGCCTACTACTACTATTCTGCAAACGAGGAATTCAGACCAGAGATGCTCCAAGG AAAGAAAGTGATTGTCACAGGGGCCAGCAAAGGGATCGGAAGAGAGATGGCTTATCATCT GGCGAAGATGGGAGCCCATGTGGTGGTGACAGCGAGCTCAGCACACTACATTGCTGGCACCATGGAAGACATGACCTTCGGAGAGCAATTTGTTGCCCAAGCAGGAAAGCTCATGGGAGG ACTAGACATGCTCATTCTCAACCACATCACCAACACTTCTTTGAATCTTTTTCATGATGA TATTCACCATGTGCGCAAAAGCATGGAAGTCAACTTCCTCAGTTACGTGGTCCTGACTGT AGCTGCCTTGCCCATGCTGAAGCAGAGCAATGGAAGCATGTGCGCTCTTCTGCTGGAATGCTATCATGTTGTGCATCTGAGCAGTNGTTGATGGTCTCTCTCATAGAAGATATCAGGCAG GCATGATATACTTTGGTCTGCTATACCAGACGCTAGGCGTCTGATGCA SEQ ID 14: NM_005525_T3 | LENGTH: 1394 GCACTGCCTGAGACTACTCCAGCCTCCCCCGTCCCTGATGTCACAATTCAGAGGCTGCTGCCTGCTTAGGAGGTTGTAGAAAGCTCTGTAGGTTCTCTCTGTGTGTCCTACAGGAGTCTT CAGGCCAGCTCCCTGTCGGATGGCTTTTATGAAAAAATATCTCCTCCCCATTCTGGGGCT CTTCATGGCCTACTACTACTATTCTGCAAACGAGGAATTCAGACCAGAGATGCTCCAAGG AAAGAAAGTGATTGTCACAGGGGCCAGCAAAGGGATCGGAAGAGAGATGGCTTATCATCTGGCGAAGATGGGAGCCCATGTGGTGGTGACAGCGAGGTCAAAAGAAACTCTACAGAAGGT GGTATCCCACTGCCTGGAGCTTGGAGCAGCCTCAGCACACTACATTGCTGGCACCATGGA AGACATGACCTTCGCAGAGCAATTTGTTGCCCAAGCAGGAAAGCTCATGGGAGGACTAGA CATGCTCATTCTCAACCACATCACCAACACTTCTTTGAATCTTTTTCATGATGATATTCACCATGTGCGCAAAAGCATGGAAGTCAACTTCCTCAGTTACGTGGTCCTGACTGTAGCTGC CTTGCCCATGCTGAAGCAGAGCAATGGAAGCATTGTTGTCGTCTCCTCTCTGGCTGGGAA AGTGGCTTATCCAATGGTTGCTGCCTATTCTGCAAGCAAGTTTGCTTTGGATGGGTTCTT CTCCTCCATCAGAAAGGAATATTCAGTGTCCAGGGTCAATGTATCAATCACTCTCTGTGTTCTTGGCCTCATAGACACAGAAACAGCCATGAAGGCAGTTTCTGGGATAGTCCATATGCA AGCAGCTCCAAAGGAGGAATGTGCCCTGGAGATCATCAAAGGGGGAGCTCTGCGCCAAGA AGAAGTGTATTATGACAGCTCACTCTGGACCACTCTTCTGATCAGAAATCCATGCAGGAA GATCCTGGAATTTCTCTACTCAACGAGCTATAATATGGAGGGACTGTTCTGTCTCATGTTTATCTGAGCTCTTATCTATGAAGACATCTTCCCAGAGTGTCCCCAGAGACATGCAAGTCA TGGGTCACACCTGACAAATGGAAGGAGTTCCTCTAACATTTGCAAAATGGAAATGTAATA ATAATGAATGTCATGCACCGCTGCAGCCAGCAGTTGTAAAATTGTTAGTAAACATAGGTA TAATTACCAGATAGTTATATTAAATTTATATCTTATATATAATAATATGTGATGATTAATACAATATTAATTATAATAAAGGTCACATAAACTTTATAAATTCATAACTGGTAGCTATAA CTTGAGCTTATTCAGGATGGTTTCTTTAAAACCATAAACTGTACAAATGAAATTTTTCAA TATTTGTTTCTTAT SEQ ID 15: NM_005525_T4 | LENGTH: 1394 GCACTGCCTGAGACTACTCCAGCCTCCCCCGTCCCTGATGTCACAATTCAGAGGCTGCTGCCTGCTTAGGAGGTTGTAGAAAGCTCTGTAGGTTCTCTCTGTGTGTCCTACAGGAGTCTT CAGGCCAGCTCCCTGTCGGATGGCTTTTATGAAAAAATATCTCCTCCCCATTCTGGGGCT CTTCATGGCCTACTACTACTATTCTGCAAACGAGGAATTCAGACCAGAGATGCTCCAAGG AAAGAAAGTGATTGTCACAGGGGCCAGCAAAGGGATCGGAAGAGAGATGGCTTATCATCTGGCGAAGATGGGAGCCCATGTGGTGGTGACAGCGAGGTCAAAAGAAACTCTACAGAAGGT GGTATCCCACTGCCTGGAGCTTGGAGCAGCCTCAGCACACTACATTGCTGGCACCATGGA AGACATGACCTTCGCAGAGCAATTTGTTGCCCAAGCAGGAAAGCTCATGGGAGGACTAGA CATGCTCATTCTCAACCACATCACCAACACTTCTTTGAATCTTTTTCATGATGATATTCACCATGTGCGCCCCATGCTGAAGCAGAGCAATGGAAGCATTGTTGTCGTCTCCTCTCTGGC TGGGAAAGTGGCTTATCCAATGGTTGCTGCCTATTCTGCAAGCAAGTTTGCTTTGGATGG GTTCTTCTCCTCCATCAGAAAGGAATATTCAGTGTCCAGGGTCAATGTATCAATCACTCT CTGTGTTCTTGGCCTCATAGACACAGAAACAGCCATGAAGGCAGTTTCTGGGATAGTCCATATGCAAGCAGCTCCAAAGGAGGAATGTGCCCTGGAGATCATCAAAGGGGGAGCTCTGCG CCAAGAAGAAGTGTATTATGACAGCTCACTCTGGACCACTCTTCTGATCAGAAATCCATG CAGGAAGATCCTGGAATTTCTCTACTCAACGAGCTATAATATGGACAGATTCATAAACAA GTAGGAACTCCCTGAGGGCTGGGCATGCTGAGGGATTTTGGGACTGTTCTGTCTCATGTTTATCTGAGCTCTTATCTATGAAGACATCTTCCCAGAGTGTCCCCAGAGACATGCAAGTCA TGGGTCACACCTGACAAATGGAAGGAGTTCCTCTAACATTTGCAAAATGGAAATGTAATA ATAATGAATGTCATGCACCGCTGCAGCCAGCAGTTGTAAAATTGTTAGTAAACATAGGTA TAATTACCAGATAGTTATATTAAATTTATATCTTATATATAATAATATGTGATGATTAATACAATATTAATTATAATAAAGGTCACATAAACTTTATAAATTCATAACTGGTAGCTATAA CTTGAGCTTATTCAGGATGGTTTCTTTAAAACCATAAACTGTACAAATGAAATTTTTCAA TATTTGTTTCTTAT SEQ ID 16: NM_005525_T5 | LENGTH: 1394 GCACTGCCTGAGACTACTCCAGCCTCCCCCGTCCCTGATGTCACAATTCAGAGCCTGCTGCCTGCTTAGGAGGTTGTAGAAAGCTCTGTAGGTTCTCTCTGTGTGTCCTACAGGAGTCTT CAGGCCAGCTCCCTGTCGGATGGCTTTTATGAAAAAATATCTCCTCCCCATTCTGGGGCT CTTCATGGCCTACTACTACTATTCTGCAAACGAGGAATTCAGACCAGAGATGCTCCAAGG AAAGAAAGTGATTGTCACAGGGGCCAGCAAAGGGATCGGAAGAGAGATGGCTTATCATCTGGCGAAGATGGGAGCCCATGTGGTGGTGACAGCGAGCTCAGCACACTACATTGCTGGCAC CATGGAAGACATGACCTTCGCAGAGCAATTTGTTGCCCAAGCAGGAAAGCTCATGGGAGG ACTAGACATGCTCATTCTCAACCACATCACCAACACTTCTTTGAATCTTTTTCATGATGA TATTCACCATGTGCGCAAAAGCATGGAAGTCAACTTCCTCAGTTACGTGGTCCTGACTGTAGCTGCCTTGCCCATGCTGAAGCAGAGCAATGGAAGCATTGTTGTCGTCTCCTCTCTGGC TGGGAAAGTGGCTTATCCAATGGTTGCTGCCTATTCTGCAAGCAAGTTTGCTTTGGATGG GTTCTTCTCCTCCATCAGAAAGGAATATTCAGTGTCCAGGGTCAATGTATCAATCACTCT CTGTGTTCTTGGCCTCATAGACACAGAAACAGCCATGAAGGCAGTTTCTGGGATAGTCCATATGCAAGCAGCTCCAAAGGAGGAATGTGCGCTGGAGATCATCAAAGGGGGAGCTCTGCG CCAAGAAGAAGTGTATTATGACAGCTCACTCTGGACCACTCTTCTGATCAGAAATCCATC CAGGAAGATCCTGGAATTTCTCTACTCAACGAGCTATAATATGGACAGATTCATAAACAA GTAGGAACTCCCTGAGGGCTGGGCATGCTGAGGGATTTTGGGACTGTTCTGTCTCATGTTTATCTGAGCTCTTATCTATGAAGACATCTTCCCAGAGTGTCCCCAGAGACATGCAAGTCA TGGGTCACACCTGACAAATGGAAGGAGTTCCTCTAACATTTGCAAAATGGAAATGTAATA ATAATGAATGTCATGCACCGCTGCAGCCAGCAGTTGTAAAATTGTTAGTAAACATAGGTA TAATTACCAGATAGTTATATTAAATTTATATCTTATATATAATAATATGTGATGATTAATACAATATTAATTATAATAAAGGTCACATAAACTTTATAAATTCATAACTGGTAGCTATAA CTTGAGCTTATTCAGGATGGTTTCTTTAAAACCATAAACTGTACAAATGAAATTTTTCAA TATTTGTTTCTTAT SEQ ID 17: NM_005525_T6 | LENGTH: 1821 GGTGAAAAGGGAAAACCTGCCCAAATCCAGTTTTTGTTTCAGTAACTTCCTTTGAGACAAAGTCAGGAATCTGAGAGTAAGCACCTGCTAAGGGTGGGACAGGGGCTCTGTCTGGTATGC CTCTCCCATGTTAAGAGCTAACAATAGTAATGGATAAGTCTCCAGGGCAACCAGGACCAC TTCCAAGCATTCCTGTCTTGGGCTGCCTCGAGGGCTCCTCTGTCCTTTGGGGAGTACTGA TTGATGCCTGATGCCCAGAACTGGCCCACTCTGGCTTCTCTTTGGAGCTGTCTCTGCAGGCGCCTTCTGGCTGCCAGCTCGGTCCTAGCATAAGGGACTTCTTCCTTGGCCTGGGTTTCA CCTTCTTGTATCAGGTGGCAGACCAGCTGGTTTCAGTCCCAAATCAGGTCTTCTGACTCC TCCCAGAAACCAACCAACTTCTGAGCAGGAAATCCTGCCCCTCCCCAAAGAGTGGGAAAC GCGAAAGGAAGAGAGAGATGAAACAGAAGGAAAGGCAGAGGAGGAGGGAGAGAGAGAGAAGAGAAGAAAAAGAAAAAAGAACATCAATAAAAAGAAGTCAGATTTGTTCGAAATCTTGAG AGATGCTCCAAGGAAAGAAAGTGATTGTCACAGGGGCCAGCAAAGGGATCGGAAGAGAGA TGGCTTATCATCTGGCGAAGATGGGAGCCCATGTGGTGGTGACAGCGAGGTCAAAAGAAA CTCTACAGAAGGTGGTATCCCACTGCCTGGAGCTTGGAGCAGCCTCAGCACACTACATTGCTGGCACCATGGAAGACATGACCTTCGCAGAGCAATTTGTTGCCCAAGCAGGAAAGCTCA TGGGAGGACTAGACATGCTCATTCTCAACCACATCACCAACACTTCTTTGAATCTTTTTC ATGATGATATTCACCATGTGCGCAAAAGCATGGAAGTCAACTTCCTCAGTTACGTGGTCC TGACTGTAGCTGCCTTGCCGATGCTGAAGCAGAGCAATGGAAGCATTGTTGTCGTCTCCTCTCTGGCTGGGAAAGTGGCTTATCCAATGGTTGCTGCCTATTCTGCAAGCAAGTTTGCTT TGGATGGGTTCTTCTCCTCCATCAGAAAGGAATATTCAGTGTCCAGGGTCAATGTATCAA TCACTCTCTGTGTTCTTGGCCTCATAGACACAGAAACAGCCATGAAGGCAGTTTCTGGGA TAGTCCATATGCAAGCAGCTCCAAAGGAGGAATGTGCCCTGGAGATCATCAAAGGGGGAGCTCTGCGCCAAGAAGAAGTGTATTATGACAGCTCACTCTGGACCACTCTTCTGATCAGAA ATCCATGCAGGAAGATCCTGGAATTTCTCTACTCAACGAGCTATAATATGGACAGATTCA TAAACAAGTAGGAACTCCCTGAGGGCTGGGCATGCTGAGGGATTTTGGGACTGTTCTGTC TCATGTTTATCTGAGCTCTTATCTATGAAGACATCTTCCCAGAGTGTCCCCAGAGACATGCAAGTCATGGGTCACACCTGACAAATGGAAGGAGTTCCTCTAACATTTGCAAAATGGAAA TGTAATAATAATGAATGTCATGCACCGCTGCAGCCAGCAGTTGTAAAATTGTTAGTAAAC ATAGGTATAATTACCAGATAGTTATATTAAATTTATATCTTATATATAATAATATGTGAT GATTAATACAATATTAATTATAATAAAGGTCACATAAACTTTATAAATTCATAACTGGTAGCTATAACTTGAGCTTATTCAGGATGGTTTCTTTAAAACCATAAACTGTACAAATGAAAT TTTTCAATATTTGTTTCTTAT SEQ ID 18: NM_005525_T7 | LENGTH: 1304 GCACTGCCTGAGACTACTCCAGCCTCCCCCGTCCCTGATGTCACAATTCAGAGGCTGCTG CCTGCTTAGGAGGTTGTAGAAAGCTCTGTAGGTTCTCTCTGTGTGTCCTACAGGAGTGTTCAGGCCAGCTCCCTGTCGGATGGCTTTTATGAAAAAATATCTCCTCCCCATTCTGGGGCT CTTCATGGCCTACTACTACTATTCTGCAAACGAGGAATTCAGACCAGAGATGCTCCAAGG AAAGAAAGTGATTGTCACAGGGGCCAGCAAAGGGATCGGAAGAGAGATGGCTTATCATCT GGCGAAGATGGGAGCCCATGTGGTGGTGACAGCGAGGTCAAAAGAAACTCTACAGAAGGTGGTATCCCACTGCCTGGAGCTTGGAGCAGCCTCAGCACACTACATTGCTGGCACCATGGA AGACATGACCTTCGCAGAGCAATTTGTTGCCCAAGCAGGAAAGCTCATGGGAGGACTAGA CATGCTCATTCTCAACCACATCACCAACACTTCTTTGAATCTTTTTCATGATGATATTCA CCATGTGCGCAAAAGCATGGAAGTCAACTTCCTCAGTTACGTGGTCCTGACTGTAGCTGC
CTTGCCCATGCTGAAGCAGAGCAATGGAAGCATTGTTGTCGTCTCCTCTCTGGCTGAAAC AGCCATGAAGGCAGTTTCTGGGATAGTCCATATGCAAGCAGCTCCAAAGGAGGAATGTGC CCTGGAGATCATCAAAGGGGGAGCTCTGCGCCAAGAAGAAGTGTATTATGACAGCTCACT CTGCACCACTCTTCTGATCAGAAATCCATGCAGGAAGATCCTGGAATTTCTCTACTCAACGAGCTATAATATGGACAGATTCATAAACAAGTAGGAACTCCCTGAGGGCTGGGCATGCTG AGGGATTTTGGGACTGTTCTGTCTCATGTTTATCTGAGCTCTTATCTATGAAGACATCTT CCCAGAGTGTCCCCAGAGACATGCAAGTCATGGGTCACACCTGACAAATGGAAGGAGTTC CTCTAACATTTGCAAAATGGAAATGTAATAATAATGAATGTCATGCACCGCTGCAGCCAGCAGTTGTAAAATTCTTAGTAAACATAGGTATAATTACCAGATAGTTATATTAAATTTATA TCTTATATATAATAATATGTCATGATTAATACAATATTAATTATAATAAAGGTCACATAA ACTTTATAAATTCATAACTGGTAGCTATAACTTGAGCTTATTCAGGATGGTTTCTTTAAA ACCATAAACTGTACAAATGAAATTTTTCAATATTTGTTTCTTAT SEQ ID 19: XM_110304_T1 |Length: 1307 WT ACTGTTGGCCTCTGGAWTCAGAGGCTGCTGCCTGCCTGGGAGGTTGTAGAAAGCTCTGCA GGTTTTCTTCGTGTGTCCTACAGGGCGCCCTGACCCAGGTCCCTGTTTGATGGCAGTTAT GAAAAATTACCTCCTCCCGATCCTGGTGCTCTTCCTGGCCTACTACTACTATTCTACAAATGAAGAGTTCAGACCAGAAATGCTCCAGGGAAAGAAAGTGATTGTCACTGGGGCCAGCAA AGGGATTGGAAGAGAAATGGCATATCATCTGTCAAAAATGGGAGCCCATGTGGTATTGAC TGCCAGGTCGGAGGAAGGTCTCCAGAAGGTAGTGTCTCGCTGCCTTGAACTCGGAGCAGC CTCTGCTCACTACATTGCTGGCACTATGGAAGACATGACATTTGCGGAGCAATTTATTGTCAAGGCGGGAAAGCTCATGGGCGGACTGGACATGCTTATTCTAAACCACATCACTCAGAC CTCGCTGTCTCTCTTCCATGACGACATCCACTCTGTGCGAAGAGTCATGGAGGTCAACTT CCTCAGCTACGTGGTCATGAGCACAGCCGCCTTGCCCATGCTGAAGCAGAGCAATGGCAG CATTGCCGTCATCTCCTCCTTGGCTGGGAAAATGACCCAGCCTATGATTGCTCCCTACTCTGCAAGCAAGTTTGCTCTGGATGGGTTCTTTTCCACCATTAGAACAGAACTCTACATAAC CAAGGTCAACGTGTCCATCACTCTCTGTGTCCTTGGCCTCATAGACACAGAAACAGCTAT GAAGGAAATCTCTGGGATAATTAACGCCCAAGCTTCTCCCAAGGAGGAGTGCCCCCTGGA GATCATCAAAGCCACAGCTCTACGCAAAACCGAGGTGTACTATGACAAATCGCCTTTGACTCCAATCCTGCTTGGGAACCCAGGAAGGAAGATCATGGAATTTTTTTCATTACGATATTA TAATAAGGACATGTTTGTAAGTAACTAGGAACTCCTGACCCCTCGTGAGTGGTCTTAGAA CACTCCTCCCTGATACTTCTGTAAGCCCTACCCACAAAAGTATCTTTCCAGAGATACACA AATTTTGGGGTACACCTCATCATGAGAAATTCTTGCAACACTTGCACAGTGAAAATGTAATTGTAATAAATGTCACAAACCACTTTGGGGCCTGCAGTTGTGAACTTGATTGTAACTATG GATATAACACATAGTGGTTGTATCGCCTTTACCTCACACTGAATGAAAACAATGATAACT AATGTAACATTAATATAATAAAGGTAATATCAACTTTGTAAATCCA SEQ ID 20: XM_110304_T3 | Length: 1181ACTGTTGGCCTCTGGAWTCAGAGGCTGCTGCCTGCCTGGGAGGTTGTAGAAAGCTCTGCA GGTTTTCTTCGTCTGTCCTACAGGGCGCCCTGAGCCAGGTCCCTGTTTGATGGCAGTTAT GAAAAATTACCTCCTCCCGATCCTGGTGCTCTTCCTGGCCTACTACTACTATTCTACAAA TGAAGAGTTCAGACTCCAGAAGGTAGTGTCTCGCTGCCTTGAACTCGGAGCAGCCTCTGCTCACTACATTGCTGGCACTATGGAAGACATGACATTTGCGGAGCAATTTATTGTCAAGGC GGGAAAGCTCATGGGCGGACTGGACATGCTTATTCTAAACCACATCACTCAGACCTCGCT GTCTCTCTTCCATGACGACATCCACTCTGTGCGAAGAGTCATGGAGGTCAACTTCCTCAG CTACGTGGTCATGAGCACAGCCGCCTTGCCCATGCTGAAGCAGAGCAATGGCAGCATTGCCGTCATCTCCTCCTTGGCTGGGAAAATGACCCAGCCTATGATTGCTCCCTACTCTGCAAG CAAGTTTGCTCTGGATGGGTTCTTTTCCACCATTAGAACAGAACTCTACATAACCAAGGT CAACCTCTCCATCACTCTCTGTGTCCTTGGCCTCATACACACAGAAACAGCTATGAAGGA AATCTCTGGGATAATTAACGCCCAAGCTTCTCCCAAGGAGGAGTCCGCCCTGGAGATCATCAAAGGCACAGCTCTACGCAAAAGCGAGGTGTACTATGACAAATCGCCTTTGACTCCAAT CCTGCTTGGGAACCCAGGAAGGAAGATCATGGAATTTTTTTCATTACGATATTATAATAA GGACATGTTTGTAAGTAACTAGGAACTCCTGAGCCCTGGTGAGTGGTCTTAGAACAGTCC TGCCTGATACTTCTGTAAGCCCTACCCACAAAAGTATCTTTCCAGAGATACACAAATTTTGGGGTACACCTCATCATGAGAAATTCTTGCAACACTTGCACAGTGAAAATGTAATTGTAA TAAATGTCACAAACCACTTTGGGGCCTGCAGTTGTGAACTTGATTGTAACTATGGATATA AACACATAGTGGTTGTATCGGCTTTACCTCACACTGAATGAAACAATGATAACTAATGTA ACATTAAATATAATAAAGGTAATATCAACTTTGTAAATGCA SEQ ID 21: XM_110304_T4 |Length: 845 ACTGTTGGCCTCTGGAWTCAGAGGCTGCTGCCTGCCTGGGAGGTTGTAGAAAGCTCTGCA GGTTTTCTTCGTGTGTCCTACAGGGCGCCCTGAGCCAGGTCCCTGTTTGATGGCAGTTAT GAAAAATTACCTCCTCCCGATCCTGGTGCTCTTCCTGGCCTACTACTACTATTCTACAAATGAAGAGTTCAGACCAGAAATGCTCCAGGGAAAGAAAGTGATTGTCACTGGGGCCAGCAA AGGGATTGGAAGAGAAATGGCATATCATCTGTCAAAAATGGGAGCCCATGTGGTATTGAC TGCCAGGTCGGAGGAAGGTCTCCAGAAGGTAGTGTCTCGCTGCCTTGAACTCGGAGCAGC CTCTGCTCACTACATTGCTGGCACTATGGAAGACATGACATTTGCGGAGCAATTTATTGTCAAGGCGGGAAAGCTCATGGGCGGACTGGACATGCTTATTCTAAACCACATCACTCAGAC CTCGCTGTCTCTCTTCCATGACGACATCCACTCTGTGCGAAGAGTCATGGAGGTCAACTT CCTCAGCTACGTGGTCATGAGCACAGCCGCCTTGCCCATGCTGAAGCAGAGCAATGGCAG CATTGCCGTCATCTCCTCCTTGCCTGGGGGAAGAACAGTTCCACAACAGAGAAGTCGCAGTGTTACTCCTGACTCCCGCGGCCCGTGATTAATATCACCAGCCACAGAATGGACTGGAAC CCTCTATCGATCTGGTGGGATTGGATATAACGAACATAGAATTACTCCTGAGACTACCAG AACTGAATAGTTCAAATCAAATCATGCCAGAATATCAGACAAATCCAAATGGCAAAACAG TTGCA SEQ ID 22 30 Adiponectin variants products: SEQ ID NO. 22:NP_004788_P1 | Length: 244 | Transcript: 1 WT MLLLGAVLLLLALPGHDQETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDG TPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLE TYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLY HDTN SEQ ID NO. 23: NP_004788_P2 | Length: 160 | Transcript: 2 MPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYD GSTGKFHCNIPGLYYFAYHITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNGLYADNDNDSTFTGFLLYHDTN SEQ ID NO. 24: NP_004788_P3 | Length: 153 | Transcript: 3 MLLLGAVLLLLALPGHDQETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDG TPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGALLSPTCPFALPR SSTISKTTMMAPLVNSTATFLGCTTLPTTSQSISEQ ID NO. 25: NP_004788_P4 | Length: 166 | Transcript: 4 MLLLGAVLLLLALPGHDQETTTQGPGVLLPLPKGACTGWMAGIPGHPGHNGAPGRDGRDG TPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGEPGEGAYVYRSAFSVGLE TYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYLNRLSSLP SEQ ID NO. 26: NP_033735_P1| Length: 247 | Transcript: 1 WT MLLLQALLFLLILPSHAEDDVTTTEELAPALVPPPKGTCAGWMAGIPGHPGHNGTPGRDG RDGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPRGFPGTPGRKGEPGEAAYMYRSAFSV GLETRVTVPNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYHITVYMKDVKVSLFKKDKAVLFTYDQYQEKNVDQASGSVLLHLEVGDQVWLQVYGDGDHNGLYADNVNDSTFTGF LLYHDTN SEQ ID NO. 27: NP_033735_P2 | Length: 160 | Transcript: 2 MTGAEGPRGFPGTPGRKGEPGEAAYVYRSAFSVGLETRVTVPNVPIRFTKIFYNQQNHYD GSTGKFYCNIPGLYYFSYHITVYMKDVKVSLFKKDKAVLFTYDQYQEKNVDQASGSVLLHLEVGDQVWLQVYGDGDHNGLYADNVNDSTFTGFLLYHDTN SEQ ID NO. 28: NP_033735_P3 | Length: 156 | Transcript: 3 MLLLQALLFLLILPSHAEDDVTTTEELAPALVPPPKGTCAGWMAGIPGHPGHNGTPGRDG RDGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPRGFPGTPGRKGEPGEAASLFPMYPFALLRSSTTNRIIMTAALASSTATFRDSTTSLTTSRCT SEQ ID NO. 29: NP_033735_P4 | Length: 169 | Transcript: 4 MLLLQALLFLLILPSHAEDDVTTTEELAPALVPPPKGTCAGWMAGIPGHPGHNGTPGRDG RDGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPRGFPGTPGRKGEPGEAAYVYRSAFSVGLETRVTVPNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYIYWLSSLP SEQ ID NO. 30: NP_033735_P5 | Length: 76 | Transcript: 5 MLLLQALLFLLILPSHAEDDVTTTEELAPALVPPPKGTCAGWMAGIPGHPGHIKIKFEGH PPGRLNCAKIWHFLQD SEQ ID 31 32 Ghrelin variants: SEQ ID NO. 31: NP_057446 | Length: 117| Transcript: 1 WT MPSPGTVCSLLLLGMLWLDLAMAGSSFLSPEHQRVQQRKESKKPPAKLQPRALAGWLRPE DGGQAEGAEDELEVRFNAPFDVGIKLSGVQYQQHSQALGKFLQDILWEEAKEAPADK SEQ ID NO. 32: NP_057446 | Length: 117 | Transcript: 2 MPSPGTVCSLLLLGMLWLDLAMAGSSFLSPEHQRVQVRPPHKAPHVVPALPLSNQLCDLEQQRHWASVFSQSTKDSGSDLTVSGRTWGLRVLNRLFPPSSRERSRRSHQPSCSPEL SEQ ID 33 42 HSD11B variants: SEQ ID NO. 33: NP_005516 | Length: 292 | Transcript: 1 WT MAFMKKYLLPILGLFMAYYYYSANEEFRPEMLQGKKVIVTGASKGIGREMAYHLAKMGAHVVVTARSKETLQKVVSHCLELGAASAHYIAGTMEDMTFAEQFVAQAGKLMGGLDMLILNH ITNTSLNLFHDDIHHVRKSMEVNFLSYVVLTVAALPMLKQSNGSIVVVSSLAGKVAYPMV AAYSASKFALDGPFSSIRKEYSVSRVNVSITLCVLGLIDTETAMKAVSGIVHMQAAPKEE CALEIIKGGALRQEEVYYDSSLWTTLLIRNPCRKILEFLYSTSYNMDRFINK SEQ ID NO. 34:NP_005516 | Length: 163 | Transcript: 2 MAFMKKYLLPILGLFMAYYYYSANEEFRPEMLQGKKVIVTGASKGIGREMAYHLAKMGAH VVVTASSAHYIAGTMEDMTFAEQFVAQAGKLMGGLDMLILNHITNTSLNLFEDDIHHVRK SMEVNFLSYVVLTVAALPMLKQSNGSMCALLLECYHVVHLSSX SEQ ID NO. 35: NP_005516 | Length: 295 |Transcript: 3 MAFNKKYLLPILGLFMAYYYYSANEEFRPEMLQGKKVIVTGASKGIGREMAYHLAKMGAH VVVTARSKETLQKVVSHCLELGAASAHYIAGTMEDMTFAEQFVAQAGKLMGGLDMLILNH ITNTSLNLFHDDIHHVRKSMEVNFLSYVVLTVAALPMLKQSNGSIVVVSSLAGKVAYPMVAAYSASKFALDGFFSSIRKEYSVSRVNVSITLCVLGLIDTETAMKAVSGIVHMQAAPKEE CALEIIKGGALRQEEVYYDSSLWTTLLIRNPCRKILEFLYSTSYNMEGLFCLMFI
SEQ ID NO. 36: NP_005516 | Length: 274 | Transcript: 4 MAFMKKYLLPILGLFMAYYYYSANEEFRPEMLQGKKVIVTGASKGIGREMAYHLAKMGAH VVVTARSKETLQKVVSHCLELGAASAHYIAGTMEDMTFAEQFVAQAGKLMGGLDMLILNH ITNTSLNLFHDDIHHVRPMLKQSNGSIVVVSSLAGKVAYPMVAAYSASKFALDGFFSSIRKEYSVSRVNVSITLCVLGLIDTETAMKAVSGIVHMQAAPKEECALEIIKGGALRQEEVYY DSSLWTTLLIRNPCRKILEFLYSTSYNMDRFINK SEQ ID NO. 37: NP_005516 | Length: 274 | Transcript: 5 MAFMKKYLLPILGLFMAYYYYSANEEFRPEMLQGKKVIVTGASKGIGREMAYHLAKMGAHVVVTASSAHYIAGTMEDMTFAEQFVAQAGKLMGGLDMLILNHITNTSLNLFHDDIHHVRK SMEVNFLSYVVLTVAALPMLKQSNGSIVVVSSLAGKVAYPMVAAYSASKFALDGFFSSIR KEYSVSRVNVSITLCVLGLIDTETAMKAVSGIVHMQAAPKEECALEIIKGGALRQEEVYY DSSLWTTLLIRNPCRKILEFLYSTSYNMDRFINK SEQ ID NO. 38: NP_005516 | Length:262 | Transcript: 6 MLQGKKVIVTGASKGIGREMAYHLAKMGAHVVVTARSKETLQKVVSHCLELGAASAHYIA GTMEDMTFAEQFVAQAGKLMGGLDMLILNHITNTSLNLFHDDIHHVRKSMEVNFLSYVVL TVAALPMLKQSNGSIVVVSSLAGKVAYPMVAAYSASKFALDGFFSSIRKEYSVSRVNVSITLCVLGLIDTETAMKAVSGIVHMQAAPKEECALEIIKGGALRQEEVYYDSSLWTTLLIRN PCRKILEFLYSTSYNMDRFINK SEQ ID NO. 39: NP_005516 | Length: 244 | Transcript: 7 MAFMKKYLLPILGLFMAYYYYSANEEFRPEMLQGKKVIVTGASKGIGREMAYHLAKMGAHVVVTARSKETLQKVVSHCLELGAASAHYIAGTMEDMTFAEQFVAQAGKLMGGLDMLILNH ITNTSLNLFHDDIHHVRKSMEVNFLSYVVLTVAALPMLKQSNGSIVVVSSLAETAMKAVS GIVHMQAAPKEECALEIIKGGALRQEEVYYDSSLWTTLLIRNPCRKILEFLYSTSYNMDR FINK SEQ ID NO. 40: XP_110304| Length: 292 | Transcript: 1 WTMAVMKNYLLPILVLFLAYYYYSTNEEFRPEMLQGKKVIVTGASKGIGREMAYHLSKMGAH VVLTARSEEGLQKVVSRCLELGAASAHYIAGTMEDMTFAEQFIVKAGKLMGGLDMLILNH ITQTSLSLFHDDIHSVRRVMEVNFLSYVVMSTAALPMLKQSNGSIAVISSLAGKMTQPMI APYSASKFALDGFFSTIRTELYITKVNVSITLCVLGLIDTETAMKEISGIINAQASPKEECALEIIKGTALRKSEVYYDKSPLTPILLGNPGRKIMEFFSLRYYNKDMFVSN SEQ ID NO. 41: XP_110304| Length: 250 | Transcript: 8 MAVMKNYLLPILVLFLAYYYYSTNEEFRLQKVVSRCLELGAASAHYIAGTMEDMTFAEQF IVKAGKLMGGLDMLILNHITQTSLSLFHDDIHSVRRVMEVNFLSYVVMSTAALPMLKQSNGSIAVISSLAGKMTQPMIAPYSASKFALDGFFSTIRTELYITKVNVSITLCVLGLIDTET AMKEISGIINAQASPKEECALEIIKGTALRKSEVYYDKSPLTPILLGNPGRKIMEFFSLR YYNKDMFVSN SEQ ID NO. 42: XP_110304| Length: 192 | Transcript: 9 MAVMKNYLLPILVLFLAYYYYSTNEEFRPEMLQGKKVIVTGASKGIGREMAYHLSKMGAHVVLTARSEEGLQKVVSRCLELGAASAHYIAGTMEDMTFAEQFIVKAGKLMGGLDMLILNH ITQTSLSLFHDDIHSVRRVMEVNFLSYVVMSTAALPMLKQSNGSIAVISSLAGGRTVPQQ RSRSVTPDSRGP
"obesity and/or diabetes Variants products--also referred at times as the "obesity and/or diabetes variants proteins" or "obesity and/or diabetes variants polypeptides"--is an amino acid sequence encoded by the obesity and/or diabetes variantsnucleic acid sequences which is a naturally occurring mRNA sequence obtained as a result of alternative splicing. The amino acid sequences may be a peptide, a protein, as well as peptides or proteins having chemically modified amino acids (see below)such as a glycopeptide or glycoprotein. The obesity and/or diabetes variants products are shown in any one of SEQ ID NO: 22 to SEQ ID NO: 42. The term also includes homologs (see below) of said sequences in which one or more amino acids has been added,deleted, substituted (see below) or chemically modified (see below) as well as fragments (see below) of this sequence having at least 10 amino acids.
"Fragments of obesity and/or diabetes related variants nucleic acid sequences"--a partial sequence of any one of SEQ ID NO:1 to SEQ ID NO:21 which includes the regions which contains the variation in nucleotides between the variant and theoriginal sequences. These regions (in the amino acid level) are as depicted in the above Table 1.
"Fragments of obesity and/or diabetes related variant product"--amino acid sequences coded by the above nucleic acid fragment, containing regions by which the variant differs from the original sequence as indicated in Table 1.
"Nucleic acid sequence"--a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may includes natural nucleotides, chemically modified nucleotides and synthetic nucleotides.
"Amino acid sequence"--a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified (see below), or composed of synthetic amino acids.
"Homologues of variants/products"--amino acid sequences of variants in which one or more amino acids has been added, deleted or replaced. The altered amino acid shall be in regions where the variant differs from the original sequence, forexample, according to the explanation in Table 1.
"Conservative substitution"--refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies inhomologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro,Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution.
"Non-conservative substitution"--refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class H residue, with a class III residue such as Asp, Asn, Glu, or Gln.
"Chemically modified"--when referring to the product of the invention, means a product (protein) where at least one of its amino acid resides is modified either by natural processes, such as processing or other post-translational modifications,or by chemical modification techniques which are well known in the art. Among the numerous known modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation,covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
"Biologically active"--refers to the variant product having some sort of biological activity, for example, capability of binding to the obesity and/or diabetes related gene or to other agonists of the original obesity and/or diabetes related geneas known.
"Immunologically active" defines the capability of a natural, recombinant or synthetic varient product, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies. Thus, forexample, an immunologically active fragment of variant product denotes a fragment which retains some or all of the immunological properties of the variant product, e.g can bind specific anti-variant product antibodies or which can elicit an immuneresponse which will generate such antibodies or cause proliferation of specific immune cells which produce variant.
"Optimal alignment"--is defined as an alignment giving the highest percent identity score. Such alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program LALIGN using aktup of 1, default parameters and the default PAM. A preferred alignment is the one performed using the CLUSTAL-W program from MacVector.TM., operated with an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix. Ifa gap needs to be inserted into a first sequence to optimally align it with a second sequence, the percent identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not considerresidues in the second sequences that are in the "gap" of the first sequence). In case of alignments of known gene sequences with that of the new variant, the optimal alignment invariably included aligning the identical parts of both sequences together,then keeping apart and unaligned the sections of the sequences that differ one from the other.
"Having at least 90% identity"--with respect to two amino acid or nucleic acid sequence sequences, refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 90% amino acidsequence identity means that 90% of the amino acids in two or more optimally aligned polypeptide sequences are identical.
"Isolated nucleic acid molecule having an variant nucleic acid sequence"--is a nucleic acid molecule that includes the obesity and/or diabetes related variant nucleic acid coding sequence. Said isolated nucleic acid molecule may include theobesity and/or diabetes related variant nucleic acid sequence as an independent insert; may include the obesity and/or diabetes related variant nucleic acid sequence fused to an additional coding sequences, encoding together a fusion protein in which thevariant coding sequence is the dominant coding sequence (for example, the additional coding sequence may code for a signal peptide); the obesity and/or diabetes related variant nucleic acid sequence may be in combination with non-coding sequences, e.g.,introns or control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host; or may be a vector in which the obesity and/or diabetes related variantprotein coding sequence is a heterologous.
"Expression vector"--refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection ofappropriate expression vectors is within the knowledge of those having skill in the art.
"Deletion"--is a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
"Insertion" or "addition"--is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.
"Substitution"--replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. As regards amino acid sequences the substitution may be conservative or non-conservative.
"Antibody"--refers to IgG, IgM, IgD, IgA, and IgG antibody. The definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of the antibodies comprising the antigen-binding domain of theanti-variant product antibodies, e.g. antibodies without the Fc portion, single chain antibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc.
"Treating a disease"--refers to administering a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring.
"Detection"--refers to a method of detection of a disease, disorder, pathological or normal condition. This term may refer to detection of a predisposition to a disease as well as for establishing the prognosis of the patient by determining theseverity of the disease.
"Probe"--the obesity and/or diabetes variant nucleic acid sequence, or a sequence complementary therewith, when used to detect presence of other similar sequences in a sample or of sequences having some homology with this sequence. The detectionis carried out by identification of hybridization complexes between the probe and the assayed sequence. The probe may be attached to a solid support or to a detectable label.
"Original obesity and/or diabetes related genes"--the amino acid or nucleic acid sequence from which the obesity and/or diabetes related variants of the invention have been varied as a result of alternative slicing. The original nucleic sequenceis the sequence of the human obesity and/or diabetes related gene depicted as SEQ ID NO: 1 for human Adiponectin and the original amino acid sequence is the sequence encoded by it; SEQ ID NO: 5 for mouse Adiponectin and the original amino acid sequenceis the sequence encoded by it; SEQ ID NO: 10 for Ghrelin and the original amino acid sequence is the sequence encoded by it; SEQ ID NO: 12 for human 11-beta-HSD and the original amino acid sequence is the sequence encoded by it; SEQ ID NO: 19 for mouse11-beta-HSD and the original amino acid sequence is the sequence encoded by it.
SUMMARY OF THE INVENTION
The present invention relates to isolated nucleic acid molecules having a sequence selected from the group consisting of: SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21 and fragments thereof comprising at least 20 nucleotides. The present inventionrelates to isolated nucleic acid molecules comprising SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21 and isolated nucleic acid molecules comprising fragments of SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21 comprising at least 20 nucleotides.
The present invention relates to PCR primers which can amplify products using sequences of SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21 as templates.
The present invention relates to methods of screening, diagnosing and monitoring individuals for obesity and/or diabetes. The methods comprise detecting the presence, absence, or quantity of a transcription product that comprises a sequenceselected from the group consisting of: SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21 in a sample. The presence or quantity of said transcription product is indicative of obesity and/or diabetes.
The present invention relates to methods of screening, diagnosing and monitoring individuals for obesity and/or diabetes comprising the step of detecting the presence, absence, or quantity of a translation product of a transcript having asequence selected from the group consisting of: SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21 in a sample. The presence or quantity of said translation product is indicative of obesity and/or diabetes
The present invention relates to kits for screening, diagnosing and monitoring an individual for obesity and/or diabetes.
The present invention relates to proteins encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21 and immunogenic fragments thereof.
The present invention relates to antibodies which specifically bind to an epitope on a protein encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21.
The present invention relates to antibodies which specifically bind to an epitope on a protein encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21 that are linked to detectable labels oractive agents.
The present invention relates to pharmaceutical composition comprising antibodies which specifically bind to an epitope on a protein encoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2 4; 6 9; 11; 13 18; 20-21that are linked to active agents.
The present invention relates to methods of treating an individual suspected of suffering from obesity and/or diabetes. The methods comprise the step of administering to individuals antibodies which specifically bind to an epitope on a proteinencoded by a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 2 4; 6 9; 11; 13 18; 20 21 that are linked to active agents.
The present invention relates to methods of delivering a nucleic acid molecule to obesity and/or diabetic cell of an individual. The methods comprise the step of administering to said individual a pharmaceutical composition comprising antibodieswhich specifically bind to an epitope on a protein encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2 4; 6 9; 11; 13-18; 20 21 and, a nucleic acid molecules.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows multiple alignment of four amino acid sequences ID NOS: 22 25 of human origin (depicted in SEQ ID NO:22 to SEQ ID NO:25 to each other and to the original sequence;
FIG. 2 shows multiple alignment of five amino acid sequences ID NOS: 26 30 of mouse origin (depicted in SEQ ID NO:26 to SEQ ID NO:30 to each other and to the original sequence;
FIG. 3 shows alignment of two amino acid sequences ID NOS: 31 32 of human origin (depicted in SEQ ID NO:31 to SEQ ID NO:32 to the original sequence;
FIG. 4 shows multiple alignment of seven amino acid sequences ID NOS: 33 39 of human origin (depicted in SEQ ID NO:33 to SEQ ID NO:39 to each other and to the original sequence;
FIG. 5 shows multiple alignment of three amino acid sequences ID NOS: 40 42 of human origin (depicted in SEQ ID NO:40 to SEQ ID NO:42 to each other and to the original sequence;
FIG. 6 shows multiple alignment of four nucleic acid sequences ID NOS: 1 4 of human origin (depicted in SEQ ID NO:1 to SEQ ID NO:4 to each other and to the original sequence;
FIG. 7 shows multiple alignment of five nucleic acid sequences ID NOS: 5 9 of mouse origin (depicted in SEQ ID NO:5 to SEQ ID NO:9 to each other and to the original sequence;
FIG. 8 shows alignment of two nucleic acid sequences ID NOS: 10 11 of human origin (depicted in SEQ ID NO:10 to SEQ ID NO:11 to the original sequence;
FIG. 9 shows multiple alignment of seven nucleic acid sequences ID NOS: 12 18 of human origin (depicted in SEQ ID NO:12 to SEQ ID NO:18 to each other and to the original sequence;
FIG. 10 shows multiple alignment of three amino acid sequences ID NOS: 19 21 of human origin (depicted in SEQ ID NO:19 to SEQ ID NO:21 to each other and to the original sequence;
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE I
Obesity and/or Diabetes Variants Nucleic Acid Sequence
The nucleic acid sequences of the invention include nucleic acid sequences which encode Obesity and/or diabetes variants products and fragments and analogs thereof. The nucleic acid sequences may alternatively be sequences complementary to theabove coding sequences, or to regions of said coding sequence. The length of the complementary sequences is sufficient to avoid the expression of the coding sequence. The nucleic acid sequences may be in the form of RNA or in the form of DNA, andinclude messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or the non-coding (anti-sense, complementary) strand. The nucleic acid sequences mayalso both include dNTPs, rNTPs as well as non naturally occurring sequences. The sequence may also be a part of a hybrid between an amino acid sequence and a nucleic acid sequence.
In a general embodiment, the nucleic acid sequence has at least 90%, identity with any one of the sequence identified as SEQ ID NO:2 to SEQ ID NO:4 or SEQ ID NO:6 to SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 to SEQ ID:18 or SEQ ID NO:20 to SEQID 21.
The nucleic acid sequences may include the coding sequence by itself. By another alternative the coding region may be in combination with additional coding sequences, such as those coding for fusion protein or signal peptides, in combinationwith non-coding sequences, such as introns and control elements, promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host, and/or in a vector or host environment in whichthe variant nucleic acid sequences is introduced as a heterologous sequence.
The nucleic acid sequences of the present invention may also have the Obesity and/or diabetes variants products coding sequences fused in-frame to a marker sequence which allows for purification of the variant product. The marker sequence maybe, for example, a hexahistidine tag to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. TheHA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al. Cell37:767 (1984)).
Also included in the scope of the invention are fragments as defined above also referred to herein as oligonucleotides, typically having at least 20 bases, preferably 20 30 bases corresponding to a region of the coding-sequence nucleic acidsequence. The fragments may be used as probes, primers, and when complementary also as antisense agents, and the like, according to known methods.
As indicated above, the nucleic acid sequence may be substantially a depicted in SEQ ID NO:2 to SEQ ID NO:4 or SEQ ID NO:6 to SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 to SEQ ID:18 or SEQ ID NO:20 to SEQ ID 21 or fragments thereof or sequenceshaving at least 90% identity to the above sequence as explained above. Alternatively, due to the degenerative nature of the genetic code, the sequence may be a sequence coding for any one of the amino acid sequence of SEQ ID NO:23 to SEQ ID NO:25 or SEQID NO:27 to SEQ ID NO:30 or SEQ ID NO:32 or SEQ ID NO:34 to SEQ ID:39 or SEQ ID NO:41 to SEQ ID 42, or fragments or analogs of said amino acid sequence.
A. Preparation of Nucleic Acid Sequences
The nucleic acid sequences may be obtained by screening cDNA libraries using oligonucleotide probes which can hybridize to or PCR-amplify nucleic acid sequences which encode the Obesity and/or diabetes variants products disclosed above. cDNAlibraries prepared from a variety of tissues are commercially available and procedures for screening and isolating cDNA clones are well-known to those of skill in the art. Such techniques are described in, for example, Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Press, Plainview, N.Y. and Ausubel FM et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
The nucleic acid sequences may be extended to obtain upstream and downstream sequences such as promoters, regulatory elements, and 5' and 3' untranslated regions (UTRs). Extension of the available transcript sequence may be performed by numerousmethods known to those of skill in the art, such as PCR or primer extension (Sambrook et al., supra), or by the RACE method using, for example, the Marathon RACE kit (Clontech, Cat. # K1802-1).
Alternatively, the technique of "restriction-site" PCR (Gobinda et al. PCR Methods Applic. 2:318 22, (1993)), which uses universal primers to retrieve flanking sequence adjacent a known locus, may be employed. First, genomic DNA is amplified inthe presence of primer to a linker sequence and a primer specific to the known region. The amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of eachround of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
Inverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al., Nucleic Acids Res. 16:8186, (1988)). The primers may be designed using OLIGO(R) 4.06 Primer Analysis Software (1992;National Biosciences Inc, Plymouth, Minn.), or another appropriate program, to be 22 30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68 72.degree. C. The method uses severalrestriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
Capture PCR (Lagerstrom, M. et al., PCR Methods Applic. 1:111 19, (1991)) is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. Capture PCR also requires multiplerestriction enzyme digestions and ligations to place an engineered double-stranded sequence into a flanking part of the DNA molecule before PCR.
Another method which may be used to retrieve flanking sequences is that of Parker, J. D., et al., Nucleic Acids Res., 19:3055 60, (1991)). Additionally, one can use PCR, nested primers and PromoterFinder.TM. libraries to "walk in" genomic DNA(PromoterFinder.TM.; Clontech, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions. Preferred libraries for screening for full length cDNAs are ones that have been size-selected to includelarger cDNAs. Also, random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes.
A randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA. Genomic libraries are useful for extension into the 5' nontranslated regulatory region.
The nucleic acid sequences and oligonucleotides of the invention can also be prepared by solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined to formcontinuous sequences up to several hundred bases.
B. Use of Obesity and/or Diabetes Variants Nucleic Acid Sequences for the Production of Obesity and/or Diabetes Variants Products
In accordance with the present invention, nucleic acid sequences specified above may be used as recombinant DNA molecules that direct the expression of Obesity and/or diabetes variant products.
As will be understood by those of skill in the art, it may be advantageous to produce Obesity and/or diabetes variants product-encoding nucleotide sequences possessing codons other than those which appear in SEQ ID NO:2 to SEQ ID NO:4 or SEQ IDNO:6 to SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 to SEQ ID:18 or SEQ ID NO:20 to SEQ ID 21 which are those which naturally occur in the human genome. Codons preferred by a particular prokaryotic or eukaryotic host (Murray, E. et al Nuc Acids Res.,17:477 508, (1989)) can be selected, for example, to increase the rate of variant product expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurringsequence.
The nucleic acid sequences of the present invention can be engineered in order to alter a Obesity and/or diabetes variants products coding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning,processing and/or expression of the product. For example, alterations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codonpreference, etc.
The present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which nucleic acid sequences of theinvention have been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the constructs further comprise regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook, et al., (supra).
The present invention also relates to host cells which are genetically engineered with vectors of the invention, and the production of the product of the invention by recombinant techniques. Host cells are genetically engineered (i.e.,transduced, transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered hostcells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the expression of the variant nucleic acid sequence. The culture conditions, such as temperature, pH and thelike, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.
The nucleic acid sequences of the present invention may be included in any one of a variety of expression vectors for expressing a product. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it isreplicable and viable in the host. The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and related sub-cloning procedures are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis. Examples of such promoters include: LTR or SV40 promoter, the E. coli lac or trp promoter, thephage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vectors also contains a ribosome binding site for translation initiation, and a transcriptionterminator. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
The vectors containing the appropriate DNA sequence as described above, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. Examples of appropriateexpression hosts include: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9; animal cells such as CHO, COS, HEK 293 or Bowes melanoma; adenoviruses;plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. The invention is not limited by the host cells employed.
In bacterial systems, a number of expression vectors may be selected depending upon the use intended for the Obesity and/or diabetes variant product. For example, when large quantities of Obesity and/or diabetes variant product are needed forthe induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such asBluescript(R) (Stratagene), in which the Obesity and/or diabetes variants polypeptides coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster J. Biol. Chem. 264:5503 5509, (1989)); pET vectors (Novagen, Madison Wis.); and the like.
In the yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al., (Methods in Enzymology153:516 544, (1987)).
In cases where plant expression vectors are used, the expression of a sequence encoding variant products may be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al.,Nature 310:511 514. (1984)) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al., EMBO J., 6:307 311, (1987)). Alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., EMBO J. 3:16711680, (1984); Broglie et al., Science 224:838 843, (1984)); or heat shock promoters (Winter J and Sinibaldi R. M., Results Probl. Cell Differ., 17:85 105, (1991)) may be used. These constructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. For reviews of such techniques, see Hobbs S. or Murry L. E. (1992) in McGraw Hill Yearbook of Science and Technology, McGraw Hill, New York, N.Y., pp 191 196; or Weissbach and Weissbach (1988) Methodsfor Plant Molecular Biology, Academic Press, New York, N.Y., pp 421 463.
Obesity and/or diabetes variants products may also be expressed in an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or inTrichoplusia larvae. The Obesity and/or diabetes variants products coding sequence may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of Obesityand/or diabetes coding sequences will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which variant protein isexpressed (Smith et al., J. Virol. 46:584, (1983); Engelhard, E. K. et al., Proc. Nat. Acad. Sci. 91:3224 7, (1994)).
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, Obesity and/or diabetes variants products coding sequences may be ligated into an adenovirustranscription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential E1 or E3 region of the viral genome will result in a viable virus capable of expressing variant protein in infected host cells(Logan and Shenk, Proc. Natl. Acad. Sci. 81:3655 59, (1984). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
Specific initiation signals may also be required for efficient translation of variants products coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where Obesity and/or diabetes variants productscoding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted,exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements andinitiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf, D. et al., (1994) Results Probl. Cell Differ., 20:125 62,(1994); Bittner et al., Methods in Enzymol 153:516 544, (1987)).
In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or thehost cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., and Battey,I. (1986) Basic Methods in Molecular Biology). Cell-free translation systems can also be employed to produce polypeptides using RNAs derived from the DNA constructs of the present invention.
A host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "pre-pro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO,HeLa, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express variant products may be transformed using expression vectors which contain viral origins of replication orendogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1 2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker isto confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate tothe cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M., et al., Cell 11:223 32, (1977)) and adenine phosphoribosyltransferase(Lowy I., et al., Cell 22:817 23, (1980)) genes which can be employed in tk- or aprt-cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler M., et al., Proc. Natl. Acad. Sci. 77:3567 70, (1980)); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al., J. Mol. Biol., 150:1 14, (1981)) and als or pat, which confer resistanceto chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilizehistinol in place of histidine (Hartman S. C. and R. C. Mulligan, Proc. Natl. Acad. Sci. 85:8047 51, (1988)). The use of visible markers has gained popularity with such markers as anthocyanins, beta-glucuronidase and its substrate, GUS, andluciferase and its substrates, luciferin and ATP, being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, C. A. et. al., MethodsMol. Biol., 55:121 131, (1995)).
Host cells transformed with nucleotide sequences encoding Obesity and/or diabetes variants products may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The product produced by arecombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing nucleic acid sequences encoding Obesity and/or diabetesvariants products can be designed with signal sequences which direct secretion of Obesity and/or diabetes variants products through a prokaryotic or eukaryotic cell membrane.
The Obesity and/or diabetes variants products may also be expressed as recombinant proteins with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are notlimited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle, Wash.). The inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and Obesity and/or diabetes variants products is useful to facilitate purification. One suchexpression vector provides for expression of a fusion protein compromising a variant polypeptide fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography, as described in Porath, et al., Protein Expression and Purification, 3:263 281, (1992)) while the enterokinase cleavage site provides a means for isolating variant polypeptide from the fusion protein. pGEX vectors (Promega,Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads(e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for anadditional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art.
The Obesity and/or diabetes variants products can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as necessary, in completingconfiguration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
Sf-9 cells were infected with Obesity and/or diabetes variants expressing baculovirus (Ac-obesity and/or diabetes variant) comprising the amino acid sequence of SEQ ID NO:23 to SEQ ID NO:25 or SEQ ID NO:27 to SEQ ID NO:30 or SEQ ID NO:32 or SEQID NO:34 to SEQ ID:39 or SEQ ID NO:41 to SEQ ID 42 at MOI of 2. The cells were grown in 28.degree. C. at continuous shaking (90 rpm). At 60 hours post infection (hpi) the medium was collected and cells were separated from the medium by centrifugationat 5000 RPM for 5 minutes. 10 ml medium was separated using cation exchange chromatography with SP-Sepharose column. The Column was equilibrated with PBS pH-6.5 and following loading of the sample on the column the column was washed with PBS to elutethe unbound proteins (flow through fraction). Elution was done with increasing concentration of NaCl at flow rate of 2 ml/min (5% NaCl/min).
The different fractions were subjected to SDS-PAGE electrophoresis and to western blotting using anti m Obesity and/or diabetes variant antibody.
C.1 Secretion:
Sf-9 cells were infected with Obesity and/or diabetes variants expressing baculovirus (Ac-obesity and/or diabetes variant) at MOI of 2. The cells were grown at 28.degree. C. at continuous shaking (90 rpm) and 1 ml samples were collected at 24,48 and 60 hours post infection (hpi). Following centrifugation Cells pellet was lysed with lysis buffer (50 mM Tris pH 7.5, 1% triton X100, and protease inhibitor cocktail) at 4.degree. C. for 30 min and sonicated for 30 seconds. The sample wascentrifuged for 10 minutes at 14000 rmp and the sup was designated Pellet. 40 .mu.l of the pellet preparation and of the medium (Designated Medium) were supplemented with sample buffer and electrophoreses on a 15% SDS-PAGE. Following electrophoresisthe gel was subjected to a semi dry protein transfer onto a nitrocellulose membrane. The membrane was incubated with anti m Obesity and/or diabetes variants antibody for 2 hours and with secondary anti rabbit antibody for an additional 1 hour.
Detection of the signal was done using a commercial western blot detection kit.
D. Diagnostic Applications Utilizing Nucleic Acid Sequences
The nucleic acid sequences of the present invention may be used for a variety of diagnostic purposes. The nucleic acid sequences may be used to detect and quantitate expression of the Obesity and/or diabetes variant in patient's cells, e.g.biopsied tissues, by detecting the presence of mRNA coding for the Obesity and/or diabetes variants products. Alternatively, the assay may be used to detect the soluble variants in the serum or blood. This assay typically involves obtaining total mRNAfrom the tissue or serum and contacting the mRNA with a nucleic acid probe. The probe is a nucleic acid molecule of at least 20 nucleotides, preferably 20 30 nucleotides, capable of specifically hybridizing with a sequence included within the sequenceof a nucleic acid molecule encoding the Obesity and/or diabetes variant product under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of variant. This assay can be used to distinguishbetween absence, presence, and excess expression of Obesity and/or diabetes variants products and to monitor levels of Obesity and/or diabetes variants expression during therapeutic intervention. In addition, the assay may be used to compare the levelsof the Obesity and/or diabetes variant of the invention to the levels of the original Obesity and/or diabetes sequence from which it has been varied or to levels of each other, which comparison may have some physiological meaning.
The invention also contemplates the use of the nucleic acid sequences as a diagnostic for diseases resulting from inherited defective variants sequences, or diseases in which the ratio of the amount of the original Obesity and/or diabetessequence from which the Obesity and/or diabetes variants were varied to the novel Obesity and/or diabetes variants of the invention is altered. These sequences can be detected by comparing the sequences of the defective (i.e., mutant) Obesity and/ordiabetes variants coding region with that of a normal coding region. Association of the sequence coding for mutant Obesity and/or diabetes variants products with abnormal variants products activity may be verified. In addition, sequences encodingmutant Obesity and/or diabetes variants products can be inserted into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, complementation experiments in a variant protein deficient strain of HEK293 cells) as yetanother means to verify or identify mutations. Once mutant genes have been identified, one can then screen populations of interest for carriers of the mutant gene.
Individuals carrying mutations in the nucleic acid sequences of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limitedto such as from blood, urine, saliva, placenta, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et al., Nature 324:163 166, (1986)) prior to analysis. RNA or cDNAmay also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention. Deletions and insertions can be detected bya change in size of the amplified product in comparison to the normal genotype.
Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such RNase and SI protection or the chemical cleavage method (e.g. Cotton, et al Proc. Natl. Acad. Sci. USA, 85:4397 4401, (1985)), or by differences in melting temperatures. "Molecular beacons" (Kostrikis L.G. et al., Science279:1228 1229, (1998)), hairpin-shaped, single-stranded synthetic oligo-nucleotides containing probe sequences which are complementary to the nucleic acid of the present invention, may also be used to detect point mutations or other sequence changes aswell as monitor expression levels of variant product. Such diagnostics would be particularly useful for prenatal testing.
Another method for detecting mutations uses two DNA probes which are designed to hybridize to adjacent regions of a target, with abutting bases, where the region of known or suspected mutation(s) is at or near the abutting bases. The two probesmay be joined at the abutting bases, e.g., in the presence of a ligase enzyme, but only if both probes are correctly base paired in the region of probe junction. The presence or absence of mutations is then detectable by the presence or absence ofligated probe.
Also suitable for detecting mutations in the Obesity and/or diabetes variants products coding sequences are oligonucleotide array methods based on sequencing by hybridization (SBH), as described, for example, in U.S. Pat. No. 5,547,839. In atypical method, the DNA target analyte is hybridized with an array of oligonucleotides formed on a microchip. The sequence of the target can then be "read" from the pattern of target binding to the array.
E. Therapeutic Applications of Nucleic Acid Sequences
Nucleic acid sequences of the invention may also be used for therapeutic purposes. Turning first to the second aspect of the invention (i.e. inhibition of expression of Obesity and/or diabetes variants), expression of Obesity and/or diabetesvariants products may be modulated through antisense technology, which controls gene expression through hybridization of complementary nucleic acid sequences, i.e. antisense DNA or RNA, to the control, 5' or regulatory regions of the gene encodingvariant product. For example, the 5' coding portion of the nucleic acid sequence sequence which codes for the product of the present invention is used to design an antisense oligonucleotide of from about 10 to 40 base pairs in length. Oligonucleotidesderived from the transcription start site, e.g. between positions -10 and +10 from the start site, are preferred. An antisense DNA oligonucleotide is designed to be complementary to a region of the nucleic acid sequence involved in transcription (Lee etal., Nucl. Acids, Res., 6:3073, (1979); Cooney et al., Science 241:456, (1988); and Dervan et al., Science251:1360, (1991)), thereby preventing transcription and the production of the variant products. An antisense RNA oligonucleotide hybridizes to themRNA in vivo and blocks translation of the mRNA molecule into the variant products (Okano J. Neurochem. 56:560, (1991)). The antisense constructs can be delivered to cells by procedures known in the art such that the antisense RNA or DNA may beexpressed in vivo. The antisense may be antisense mRNA or DNA sequence capable of coding such antisense mRNA. The antisense mRNA or the DNA coding thereof can be complementary to the full sequence of nucleic acid sequences coding for the Obesity and/ordiabetes variant protein or to a fragment of such a sequence which is sufficient to inhibit production of a protein product. Antisense technologies can also be used for inhibiting expression of one variant as compared to the other, or inhibiting theexpression of the variant/s as compared to the original sequence.
Turning now to the first aspect of the invention, i.e. expression of Obesity and/or diabetes variants, expression of Obesity and/or diabetes variants products may be increased by providing coding sequences for coding for said Obesity and/ordiabetes variants products under the control of suitable control elements ending its expression in the desired host.
The nucleic acid sequences of the invention may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier orexcipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
The products of the invention may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy." Cells from a patient may be engineered with a nucleic acidsequence (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in theart by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptides of the presentinvention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering products of the present invention by such method should be apparent to those skilled in the artfrom the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable deliveryvehicle.
Retroviruses from which the retroviral plasmid vectors mentioned above may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avianleukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, psi-2, psi-AM, PA12, T19-14X,VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller (Human Gene Therapy, Vol. 1, pg. 5 14, (1990)). The vector may transduce the packaging cells through any means known in the art. Such means include, but arenot limited to, electroporation, the use of liposomes, and CaPO.sub.4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitroor in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well ashematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
The genes introduced into cells may be placed under the control of inducible promoters, such as the radiation-inducible Egr-1 promoter, (Maceri, H. J., et al., Cancer Res., 56(19):4311 (1996)), to stimulate variant production or antisenseinhibition in response to radiation, eg., radiation therapy for treating tumors.
EXAMPLE II
Obesity and/or Diabetes Variants Products
The substantially purified Obesity and/or diabetes variant product of the invention has been defined above as the product coded from the nucleic acid sequence of the invention. Preferably the amino acid sequence is an amino acid sequence havingat least 90% identity the sequence identified as SEQ ID NO:23 to SEQ ID NO:25 or SEQ ID NO:27 to SEQ ID NO:30 or SEQ ID NO:32 or SEQ ID NO:34 to SEQ ID:39 or SEQ ID NO:41 to SEQ ID 42. The protein or polypeptide may be in mature and/or modified form,also as defined above, for example, modified by cleavage of the leader sequence. Also contemplated are protein fragments having at least 10 contiguous amino acid residues, preferably at least 10 20 residues, derived from the Obesity and/or diabetesvariant products, as well as homologues as explained above.
The sequence variations are preferably those that are considered conserved substitutions, as defined above. Thus, for example, a protein with a sequence having at least 90% sequence identity with the products identified as SEQ ID NO:23 to SEQ IDNO:25 or SEQ ID NO:27 to SEQ ID NO:30 or SEQ ID NO:32 or SEQ ID NO:34 to SEQ ID:39 or SEQ ID NO:41 to SEQ ID 42, preferably by utilizing conserved substitutions as defined above is also part of the invention, and provided that it is not identical to theoriginal peptide from which it has been varied (typically the substitutions are in regions where the variant differs from the original sequence as for example in Table 1). In a more specific embodiment, the protein has or contains the sequenceidentified SEQ ID NO:23 to SEQ ID NO:25 or SEQ ID NO:27 to SEQ ID NO:30 or SEQ ID NO:32 or SEQ ID NO:34 to SEQ ID:39 or SEQ ID NO:41 to SEQ ID 42. The Obesity and/or diabetes variants products may be (i) one in which one or more of the amino acidresidues in a sequence listed above are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) onein which the Obesity and/or diabetes variants products is fused with another compound, such as a compound to increase the half-life of the protein (for example, polyethylene glycol (PEG)), or a moiety which serves as targeting means to direct the proteinto its target tissue or target cell population (such as an antibody), or (iv) one in which additional amino acids are fused to the Obesity and/or diabetes variant product. Such fragments, variants and derivatives are deemed to be within the scope ofthose skilled in the art from the teachings herein.
A. Preparation of Obesity and/or Diabetes Variants Products
Recombinant methods for producing and isolating the Obesity and/or diabetes variant products, and fragments of the protein are described above.
In addition to recombinant production, fragments and portions of variant products may be produced by direct peptide synthesis using solid-phase techniques (cf. Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco;Merrifield J., J. Am. Chem. Soc., 85:2149 2154, (1963)). In vitro peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer. Fragments of Obesity and/or diabetes variants products may be chemically synthesized separately and combined using chemical methods to produce the full lengthmolecule.
2nd. Therapeutic Uses and Compositions Utilizing the Obesity and/or Diabetes Variants Products
The Obesity and/or diabetes variants products of the invention are generally useful in treating obesity and/or diabetes.
Obesity and/or diabetes variant products or fragments may be administered by any of a number of routes and methods designed to provide a consistent and predictable concentration of compound at the target organ or tissue. The product-containingcompositions may be administered alone or in combination with other agents, such as stabilizing compounds, and/or in combination with other pharmaceutical agents such as drugs or hormones.
Obesity and/or diabetes variants product-containing compositions may be administered by a number of routes including, but not limited to oral, intravenous, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means as well asby nasal application. Obesity and/or diabetes variant product-containing compositions may also be administered via liposomes. Such administration routes and appropriate formulations are generally known to those of skill in the art.
The Obesity and/or diabetes variants products can be given via intravenous or intraperitoneal injection. Similarly, the product may be injected to other localized regions of the body. The product may also be administered via nasal insufflation. Enteral administration is also possible. For such administration, the product should be formulated into an appropriate capsule or elixir for oral administration, or into a suppository for rectal administration.
The foregoing exemplary administration modes will likely require that the product be formulated into an appropriate carrier, including ointments, gels, suppositories. Appropriate formulations are well known to persons skilled in the art.
Dosage of the product will vary, depending upon the potency and therapeutic index of the particular polypeptide selected.
A therapeutic composition for use in the treatment method can include the product in a sterile injectable solution, the polypeptide in an oral delivery vehicle, the product in an aerosol suitable for nasal administration, or the product in anebulized form, all prepared according to well known methods. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The product of the invention may also be used to modulate endothelial differentiation and proliferation as well as to modulate apoptosis either ex vivo or in vitro, forexample, in cell cultures.
EXAMPLE III
Anti-Variant Antibodies
A. Synthesis
In still another aspect of the invention, the purified variants products are used to produce anti-variant antibodies which have diagnostic and therapeutic uses related to the activity, distribution, and expression of the Obesity and/or diabetesvariants products.
Antibodies to the Obesity and/or diabetes variant may be generated by methods well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragmentsproduced by an Fab expression library. Antibodies, i.e., those which inhibit dimer formation, are especially preferred for therapeutic use.
A fragment of the Obesity and/or diabetes variants products for antibody induction is not required to feature biological activity but has to feature immunological activity; however, the protein fragment or oligopeptide must be antigenic. Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids of the sequences specified in SEQ ID NO:23 to SEQ ID NO:25 or SEQ ID NO:27 to SEQ ID NO:30 or SEQ IDNO:32 or SEQ ID NO:34 to SEQ ID:39 or SEQ ID NO:41 to SEQ ID 42. Preferably they should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Shortstretches of Obesity and/or diabetes variants proteins amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for theproduction of antibodies to Obesity and/or diabetes variants products.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with Obesity and/or diabetes variants products or any portion, fragment or oligopeptide which retains immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants.
Monoclonal antibodies to Obesity and/or diabetes variants protein may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridomatechnique originally described by Koehler and Milstein (Nature 256:495 497, (1975)), the human B-cell hybridoma technique (Kosbor et al., Immunol. Today 4:72, (1983); Cote et al., Proc. Natl. Acad. Sci. 80:2026 2030. (1983)) and the EBV-hybridomatechnique (Cole, et al., Mol. Cell Biol. 62:109 120, (1984)).
Techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can also be used (Morrison et al.,Proc. Natl. Acad. Sci. 81:6851 6855, (1984); Neuberger et al., Nature 312:604 608, (1984); Takeda et al., Nature 314:452 454, (1985)). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) canbe adapted to produce single-chain antibodies specific for the variant protein.
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (Proc. Natl. Acad. Sci. 86:3833 3837, 1989)), and Winter G and Milstein C., (Nature 349:293 299, (1991)).
Antibody fragments which contain specific binding sites for the Obesity and/or diabetes variant protein may also be generated. For example, such fragments include, but are not limited to, the F(ab').sub.2 fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab').sub.2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity (Huse W.D. et al., Science 256:1275 1281, (1989)).
B. Diagnostic Applications of Antibodies
A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the formation of complexesbetween the Obesity and/or diabetes variants products and its specific antibody and the measurement of complex formation. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specificvariant product is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox D. E., et al., (J. Exp. Med. 158:1211, (1983)).
Antibodies which specifically bind the Obesity and/or diabetes variant product are useful for the diagnosis of conditions or diseases characterized by expression of the novel Obesity and/or diabetes variants of the invention (where normally it isnot expressed) by over or under expression of Obesity and/or diabetes variants as well as for detection of diseases in which the proportion between the amount of the Obesity and/or diabetes variants of the invention and the original Obesity and/ordiabetes sequence from which it varied is altered. Alternatively, such antibodies may be used in assays to monitor patients being treated with Obesity and/or diabetes variants products. Diagnostic assays for variants proteins include methods utilizingthe antibody and a label to detect variants products in human body fluids or extracts of cells or tissues. The products and antibodies of the present invention may be used with or without modification. Frequently, the proteins and antibodies will belabeled by joining them, either covalently or noncovalently, with a reporter molecule. A wide variety of reporter molecules are known in the art.
A variety of protocols for measuring the Obesity and/or diabetes variants products, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS). As noted. above, a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on Obesity and/or diabetes variantsproducts is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, et al. (supra). Such protocols provide a basis for diagnosing altered or abnormal levels of Obesity and/or diabetesvariants products expression. Normal or standard values for Obesity and/or diabetes variants products expression are established by combining body fluids or cell extracts taken from normal subjects, preferably human, with antibodies to Obesity and/ordiabetes variants products under conditions suitable for complex formation which are well known in the art. The amount of standard complex formation may be quantified by various methods, preferably by photometric methods. Then, standard values obtainedfrom normal samples may be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values establishes the presence of disease state.
The antibody assays are useful to determine the level of Obesity and/or diabetes variants products present in a body fluid sample, in order to determine whether it is being expressed at all, whether it is being overexpressed or underexpressed inthe tissue, or as an indication of how Obesity and/or diabetes variants levels of variable products are responding to drug treatment.
3rd. Therapeutic Uses of Antibodies
In addition to their diagnostic use the antibodies may have a therapeutical utility in blocking or decreasing the activity of the obesity and/or diabetes variants products in pathological conditions where beneficial effect can be achieved by sucha decrease.
The antibody employed is preferably a humanized monoclonal antibody, or a human Mab produced by known globulin-gene library methods. The antibody is administered typically as a sterile solution by IV injection, although other parenteral routesmay be suitable. Typically, the antibody is administered in an amount between about 1 15 mg/kg body weight of the subject. Treatment is continued, e.g., with dosing every 1 7 days, until a therapeutic improvement is seen.
Although the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention.
EXAMPLE 1
Separation
Sf-9 cells are infected with Obesity and/or diabetes variants expressing baculovirus (AC-obesity and/or diabetes variant) comprising the amino acid sequence of SEQ ID NO:23 to SEQ ID NO:25 or SEQ ID NO:27 to SEQ ID NO:30 or SEQ ID NO:32 or SEQ IDNO:34 to SEQ ID NO:39 or SEQ ID NO:41 to SEQ ID NO:42 at MOI of 2. The cells are grown in 28.degree. C. at continuous shaking (90 rpm). At 60 hours post-infection (hpi), the medium is collected and cells are separated from the medium by centrifugationat 5000 rpm for 5 minutes. 10 mL medium is separated using cation exchange chromatography with a SP-Sepharose column. The column is equilibrated with PBS pH 6.5, and, following loading of the sample on the column, the column is washed with PBS to elutethe unbound proteins (flow through fraction). Elution is done with increasing concentration of NaCl at a flow rate of 2 mL/min (5% NaCl/min).
The different fractions are subjected to SDS-PAGE electrophoresis and to western blotting using anti-Obesity and/or diabetes variant antibody.
EXAMPLE 2
Secretion
Sf-9 cells are infected with Obesity and/or diabetes variants expressing baculovirus (Ac-obesity and/or diabetes variant) at MOI of 2. The cells are grown at 28.degree. C. at continuous shaking (90 rpm), and 1 mL samples are collected at 24,48, and 60 hours post-infection (hpi). Following centrifugation, cell pellets are lysed with lysis buffer (50 mM Tris pH 7.5, 1% triton X100, and protease inhibitor cocktail) at 4.degree. C. for 30 mm and sonicated for 30 seconds. The sample iscentrifuged for 10 minutes at 14000 rpm and the supernatant is designated Pellet. 40 .mu.L of the Pellet preparation and of the medium (Designated Medium) are supplemented with sample buffer and are electrophoresed on a 15% SDS-PAGE. Followingelectrophoresis, the gel is subjected to a semi-dry protein transfer onto a nitrocellulose membrane. The membrane is incubated with anti-Obesity and/or diabetes variants antibody for 2 hours and with secondary anti-rabbit antibody for an additional 1hour.
Detection of the signal is done using a commercial western blot detection kit.
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42 DNA Homo sapiens tccat accagagggg ctcaggatgc tgttgctggg agctgttcta ctgctattag 6cccgg gcatgaccag gaaaccacgactcaagggcc cggagtcctg cttcccctgc agggggc ctgcacaggt tggatggcgg gcatcccagg gcatccgggc cataatgggg caggccg tgatggcaga gatggcaccc ctggtgagaa gggtgagaaa ggagatccag 24attgg tcctaaggga gacatcggtg aaaccggagt acccggggct gaaggtcccc 3ctttcc gggaatccaa ggcaggaaag gagaacctgg agaaggtgcc tatgtatacc 36gcatt cagtgtggga ttggagactt acgttactat ccccaacatg cccattcgct 42aagat cttctacaat cagcaaaacc actatgatgg ctccactggt aaattccact 48attcc tgggctgtac tactttgcct accacatcacagtctatatg aaggatgtga 54agcct cttcaagaag gacaaggcta tgctcttcac ctatgatcag taccaggaaa 6tgtgga ccaggcctcc ggctctgtgc tcctgcatct ggaggtgggc gaccaagtct 66caggt gtatggggaa ggagagcgta atggactcta tgctgataat gacaatgact 72ttcacaggctttctt ctctaccatg acaccaactg atcaccacta actcagagcc 78caggc caaacagccc caaagtcaat taaaggcttt cagtacggtt aggaagttga 84attta gttggaggcc tttagatatt attcattcat ttactcattc atttattcat 9tcatca agtaacttta aaaaaatcat atgctatgtt cccagtcctggggagcttca 96atgac cagataactg actagaaaga agtagttgac agtgctattt tgtgcccact ctctcctg atgctcatat caatcctata aggcacaggg aacaagcatt ctcctgtttt cagattgt atcctgaggc tgagagagtt aagtgaatgt ctaaggtcac acagtattaa gacagtgc tagaaatcaaacccagagct gtggactttg ttcactagac tgtgcccttt tagaggta catgttctct ttggagtgtt ggtaggtgtc tgtttcccac ctcacctgag ccattgaa tttgccttcc tcatgaatta aaacctcccc caagcagagc ttcctcagag agtggttc tatgatgaag tcctgtcttg gaaggactac tactcaatggcccctgcact tctacttc ctcttaccta tgtcccttct catgcctttc cctccaacgg ggaaagccaa ccatctct aagtgctgaa ctcatccctg ttcctcaagg ccacctggcc aggagcttct gatgtgat atccactttt tttttttttt gagatggagt ctcactctgt cacccaggct agtacagt gacacgacctcggctcactg cagcctcctt ctcctgggtc caagcaatta gtgcctca gcctcccgag tagctgagac ttcaggtgca ttccaccaca catggctaat ttgtattt ttagtagaaa tggggtttcg tcatgttggc caggctggtc tcgaactcct cctaggtg atccacccgc ctcgacctcc caaagtgctg ggattacaggcatgagccac tgcccagt cgatatctca ctttttattt tgccatggat gagagtcctg ggtgtgagga acctccca ccaggctaga ggcaactgcc caggaaggac tgtgcttccg tcacctctaa cccttgca gatccttgat aaatgcctca tgaagaccaa tctcttgaat cccatatcta cagaatta actccattccagtctctgca tgtaatcagt tttatccaca gaaacatttt 2tttagga aatccctggt ttaagtatca atccttgttc agctggacaa tatgaatctt 2cactgaa gttagggatg actgtgattt tcagaacacg tccagaattt ttcatcaaga 2tagcttg agcctgaaat gcaaaaccca tggaggaatt ctgaagccattgtctccttg 222caaca gggtcaggga agactgggcc tcctgaattt attattgttc tttaagaatt 228ttgag gtagttgatg gtggtaaaca ttctctcagg agacaataac tccagtgatg 234caaag attttagcaa aaacagagta aatagcattc tctatcaata tataaattta 24actatc tttttgcttacagttttaaa ttctgaacaa tttctcttat atgtgtattg 246catta aggtattatt ttttccacat ataaagcttt gtctttttgt tgttgttgtt 252taaga tggagtttcc ctctgttgcc aggctagagt gcagtggcat gatctcggct 258caacc tttgcctccc aggtttaagc gattcttctg cctcagcctcccgagtagct 264cacag gtgcctacca ccatgccagg ctaatttttg tatttttagt aaagacaggg 27accata ttggccaggc tggtctcgaa ctcctgacct tgtgatctgc ccgcctccat 276tgtta tttgtgagaa agatagatat gaggtttaga gagggatgaa gaggtgagag 282cttgt gttagtcagaactctgtgtt gtgaatgtca ttcacaacag aaaacccaaa 288atgca aactactgta agcaagaaaa ataaaggaaa aatggaaaca tttattcctt 294aatag aaattaccag agttgttctg tctttagata aggtttgaac caaagctcaa 3aatcaag acccttttct gtatgtcctt ctgttctgcc ttccgcagtgtaggctttac 3caggtgc tacacagtat agttctaggg tttccctccc gatatcaaaa agactgtggc 3cccagct ctcgtatccc caagccacac catctggcta aatggacatc atgttttctg 3atgccca aagaggagag aggaagctct ctttcccaga tgccccagca agtgtaacct 324ctcat tgctctggctgagttgtgtg cctgtttctg accaatcact gagtcaggag 33aaatat tcatattgac ttaattgcag cttaagttag gggtatgtag aggtattttc 336agcaa aattgggaca ctgttatcag aaataggaga gtggatgata gatgcaaaat 342ctgtc cacaacaaac tcttaatgct gtgtttgagc tttcatgagtttcccagaga 348agctg gaaaattcct attgattttc tctaaaattt caacaagtag ctaaagtctg 354gctca cagtctcaca tctggtgggg gtgggctcct tacagaacac gctttcacag 36cctaaa ctctctgggg cagggttatt cctttgtgga accagaggca cagagacagt 366gaggc ccaacagaggcctgagagaa actgaggtca agatttcagg attaatggtc 372atgct ttgaagtaca attgtggatt tgtccaattc tctttagttc tgtcagcttt 378catat attttagcgc tctattatta gatatataca tgtttagtat tatgtcttat 384cattt actctcttat cattatgtaa tgtccttctt tatctgtgataattttctgt 39tgaagt ctactttgtc taaaaataac atacgcactc aacttccttt tctttcttcc 396ttctt tcttccttcc tttctttctc tctctctctt tccttccttc cttcctcctt 4tctctct ctctctctct ctctcttttc ttgacagact ctcgttctgt ggccctggct 4gttcagt ggtgtgatcttggctcactg ctacctctac catgagcaat tctcctgcct 4cctccca agtagctgga actacaggct catgccactg cgcccagcta atttttgtat 42cgtaga gacggggttt caccacattc gtcaggttgg tttcaaactc ctgactttgt 426acccg cctcggcctc ccaaagtgct gggattacag gcatgagccatcacacctgg 432tttct tttgattagt gtttttgtgg tatatctttt tccatcatgt tactttaaat 438tatat tattgtattt aaaatgtgtt tcttacagac tgcatgtagt tgggtataat 444tccag tctaaaaata tctgtctttt aattggtgtt tagacaattt atatttaata 45ggtgga atttaaa 454 DNA Homo sapiens 2 atgacccggg gctgaaggtc cccgaggctt tccgggaatc caaggcagga aaggagaacc 6aaggt gcctatgtat accgctcagc attcagtgtg ggattggaga cttacgttac ccccaac atgcccattc gctttaccaa gatcttctac aatcagcaaa accactatga ctccact ggtaaattccactgcaacat tcctgggctg tactactttg cctaccacat 24tctat atgaaggatg tgaaggtcag cctcttcaag aaggacaagg ctatgctctt 3tatgat cagtaccagg aaaataatgt ggaccaggcc tccggctctg tgctcctgca 36aggtg ggcgaccaag tctggctcca ggtgtatggg gaaggagagc gtaatggact42ctgat aatgacaatg actccacctt cacaggcttt cttctctacc atgacaccaa 48484 3 7Homo sapiens 3 ctgattccat accagagggg ctcaggatgc tgttgctggg agctgttcta ctgctattag 6cccgg gcatgaccag gaaaccacga ctcaagggcc cggagtcctg cttcccctgc agggggc ctgcacaggt tggatggcgg gcatcccagg gcatccgggc cataatgggg caggccg tgatggcaga gatggcaccc ctggtgagaa gggtgagaaa ggagatccag 24attgg tcctaaggga gacatcggtg aaaccggagt acccggggct gaaggtcccc 3ctttcc gggaatccaa ggcaggaaag gagaacctggagaaggtgcg ttactatccc 36tgccc attcgcttta ccaagatctt ctacaatcag caaaaccact atgatggctc 42gtaaa ttccactgca acattcctgg gctgtactac tttgcctacc acatcacagt 48tgaag gatgtgaagg tcagcctctt caagaaggac aaggctatgc tcttcaccta 54agtaccaggaaaata atgtggacca ggcctccggc tctgtgctcc tgcatctgga 6ggcgac caagtctggc tccaggtgta tggggaagga gagcgtaatg gactctatgc 66atgac aatgactcca ccttcacagg ctttcttctc taccatgaca ccaactga 77 DNA Homo sapiens 4 ctgattccat accagagggg ctcaggatgctgttgctggg agctgttcta ctgctattag 6cccgg gcatgaccag gaaaccacga ctcaagggcc cggagtcctg cttcccctgc agggggc ctgcacaggt tggatggcgg gcatcccagg gcatccgggc cataatgggg caggccg tgatggcaga gatggcaccc ctggtgagaa gggtgagaaa ggagatccag 24attgg tcctaaggga gacatcggtg aaaccggagt acccggggct gaaggtcccc 3ctttcc gggaatccaa ggcaggaaag gagaacctgg agaaggtgcc tatgtatacc 36gcatt cagtgtggga ttggagactt acgttactat ccccaacatg cccattcgct 42aagat cttctacaat cagcaaaacc actatgatggctccactggt aaattccact 48attcc tgggctgtac cttcacaggc tttcttctct accatgacac caactga 537 5 A Mus musculus 5 atgagacctg gccactttct cctcatttct gtctgtacga ttgtcagtgg atctgacgac 6aaggg ctcaggatgc tactgttgca agctctcctg ttcctcttaa tcctgcccagtgccgaa gatgacgtta ctacaactga agagctagct cctgctttgg tccctccacc gggaact tgtgcaggtt ggatggcagg catcccagga catcctggcc acaatggcac 24gccgt gatggcagag atggcactcc tggagagaag ggagagaaag gagatgcagg 3cttggt cctaagggtg agacaggagatgttggaatg acaggagctg aagggccacg 36tcccc ggaacccctg gcaggaaagg agagcctgga gaagccgctt atgtgtatcg 42cgttc agtgtggggc tggagacccg cgtcactgtt cccaatgtac ccattcgctt 48agatc ttctacaacc aacagaatca ttatgacggc agcactggca agttctactg 54ttccg ggactctact acttctctta ccacatcacg gtgtacatga aagatgtgaa 6agcctc ttcaagaagg acaaggccgt tctcttcacc tacgaccagt atcaggaaaa 66tggac caggcctctg gctctgtgct cctccatctg gaggtgggag accaagtctg 72aggtg tatggggatg gggaccacaa tggactctatgcagataacg tcaacgactc 78ttact ggctttcttc tctaccatga taccaactga ctgcaactac ccatagccca 84cagga gaatcatgga acagtcgaca cactttcagc ttagtttgag agattgattt 9gcttag tttgagagtc ctgagtatta tccacacgtg tactcacttg ttcattaaac 96tataaaaaataattt gtgttcctag tccagaaaaa aaggcactcc ctggtctcca actcttac atggtagcaa taacagaatg aaaatcacat ttggtatggg ggcttcacaa ttcgcatg actgtctgga agtagaccat gctatttttc tgctcactgt acacaaatat ttcacata aaccctataa tgtaaatatg aaatacagtgattactcttc tcacaggctg tgtatgaa ttctaaagac ccataagtat taaagtggta gggataaatt ggaaaaaaaa aaaaaaaa agaaaaactt tagagcacac tggcggccgt tactag A Mus musculus 6 gctcattcat cttttaattc acccataaag gctttgaaaa ctaaggctgg agatgaactt 6agcct gccaggccgt ggagagtgag gaagcagaga tgacggagat gatgtctttc gtcctgt gaaatggatt gtgggtagag gttccggaga taatgcctct tgctggaaac ctgggca gttctgttcc cgccattcac agaattcttc tcactttcta ggtcttcttg 24aaggg tgagacagga gatgttggaa tgacaggagctgaagggcca cggggcttcc 3aacccc tggcaggaaa ggagagcctg gagaagccgc ttatgtgtat cgctcagcgt 36gtggg gctggagacc cgcgtcactg ttcccaatgt acccattcgc tttactaaga 42tacaa ccaacagaat cattatgacg gcagcactgg caagttctac tgcaacattc 48ctctactacttctct taccacatca cggtgtacat gaaagatgtg aaggtgagcc 54aagaa ggacaaggcc gttctcttca cctacgacca gtatcaggaa aagaatgtgg 6ggcctc tggctctgtg ctcctccatc tggaggtggg agaccaagtc tggctccagg 66gggga tggggaccac aatggactct atgcagataa cgtcaacgactctacattta 72tttct tctctaccat gataccaact gactgcaact acccatagcc catacaccag 78tcatg gaacagtcga cacactttca gcttagtttg agagattgat tttattgctt 84gagag tcctgagtat tatccacacg tgtactcact tgttcattaa acgactttat 9aataat ttgtgttcctagtccagaaa aaaaggcact ccctggtctc cacgactctt 96gtagc aataacagaa tgaaaatcac atttggtatg ggggcttcac aatattcgca actgtctg gaagtagacc atgctatttt tctgctcact gtacacaaat attgttcaca aaccctat aatgtaaata tgaaatacag tgattactct tctcacaggctgagtgtatg ttctaaag acccataagt attaaagtgg tagggataaa ttgg A Mus musculus 7 atgagacctg gccactttct cctcatttct gtctgtacga ttgtcagtgg atctgacgac 6aaggg ctcaggatgc tactgttgca agctctcctg ttcctcttaa tcctgcccag tgccgaagatgacgtta ctacaactga agagctagct cctgctttgg tccctccacc gggaact tgtgcaggtt ggatggcagg catcccagga catcctggcc acaatggcac 24gccgt gatggcagag atggcactcc tggagagaag ggagagaaag gagatgcagg 3cttggt cctaagggtg agacaggaga tgttggaatg acaggagctgaagggccacg 36tcccc ggaacccctg gcaggaaagg agagcctgga gaagccgcgt cactgttccc 42accca ttcgctttac taagatcttc tacaaccaac agaatcatta tgacggcagc 48caagt tctactgcaa cattccggga ctctactact tctcttacca catcacggtg 54gaaag atgtgaaggtgagcctcttc aagaaggaca aggccgttct cttcacctac 6agtatc aggaaaagaa tgtggaccag gcctctggct ctgtgctcct ccatctggag 66agacc aagtctggct ccaggtgtat ggggatgggg accacaatgg actctatgca 72cgtca acgactctac atttactggc tttcttctct accatgatac caactgactg78accca tagcccatac accaggagaa tcatggaaca gtcgacacac tttcagctta 84agaga ttgattttat tgcttagttt gagagtcctg agtattatcc acacgtgtac 9ttgttc attaaacgac tttataaaaa ataatttgtg ttcctagtcc agaaaaaaag 96ccctg gtctccacga ctcttacatggtagcaataa cagaatgaaa atcacatttg atgggggc ttcacaatat tcgcatgact gtctggaagt agaccatgct atttttctgc actgtaca caaatattgt tcacataaac cctataatgt aaatatgaaa tacagtgatt tcttctca caggctgagt gtatgaattc taaagaccca taagtattaa agtggtaggg aaattgg A Mus musculus 8 atgagacctg gccactttct cctcatttct gtctgtacga ttgtcagtgg atctgacgac 6aaggg ctcaggatgc tactgttgca agctctcctg ttcctcttaa tcctgcccag tgccgaa gatgacgtta ctacaactga agagctagct cctgctttgg tccctccacc gggaact tgtgcaggtt ggatggcagg catcccagga catcctggcc acaatggcac 24gccgt gatggcagag atggcactcc tggagagaag ggagagaaag gagatgcagg 3cttggt cctaagggtg agacaggaga tgttggaatg acaggagctg aagggccacg 36tcccc ggaacccctg gcaggaaagg agagcctggagaagccgctt atgtgtatcg 42cgttc agtgtggggc tggagacccg cgtcactgtt cccaatgtac ccattcgctt 48agatc ttctacaacc aacagaatca ttatgacggc agcactggca agttctactg 54ttccg ggactctaca tttactggct ttcttctcta ccatgatacc aactgactgc 6acccatagcccataca ccaggagaat catggaacag tcgacacact ttcagcttag 66gagat tgattttatt gcttagtttg agagtcctga gtattatcca cacgtgtact 72gttca ttaaacgact ttataaaaaa taatttgtgt tcctagtcca gaaaaaaagg 78cctgg tctccacgac tcttacatgg tagcaataac agaatgaaaatcacatttgg 84gggct tcacaatatt cgcatgactg tctggaagta gaccatgcta tttttctgct 9gtacac aaatattgtt cacataaacc ctataatgta aatatgaaat acagtgatta 96ctcac aggctgagtg tatgaattct aaagacccat aagtattaaa gtggtaggga aattgg 3Musmusculus 9 atgagacctg gccactttct cctcatttct gtctgtacga ttgtcagtgg atctgacgac 6aaggg ctcaggatgc tactgttgca agctctcctg ttcctcttaa tcctgcccag tgccgaa gatgacgtta ctacaactga agagctagct cctgctttgg tccctccacc gggaact tgtgcaggtt ggatggcaggcatcccagga catcctggcc acataaaaat 24tcgag gggcatccac caggccggct gaattgtgcc aaaatatggc acttcctgca 3aa 365 DNA Homo sapiens tggatg ggtgctgttt agacaaacgc cgtctcctat ataagacctg acagcacagg 6ctccg ccaggactgc aggcccacctgtctgcaacc cagctgaggc catgccctcc gggaccg tctgcagcct cctgctcctc ggcatgctct ggctggactt ggccatggca tccagct tcctgagccc tgaacaccag agagtccagc agagaaagga gtcgaagaag 24agcca agctgcagcc ccgagctcta gcaggctggc tccgcccgga agatggaggt 3cagaag gggcagagga tgaactggaa gtccggttca acgccccctt tgatgttgga 36gctgt caggggttca gtaccagcag cacagccagg ccctggggaa gtttcttcag 42cctct gggaagaggc caaagaggcc ccagccgaca agtgatcgcc cacaagcctt 48cctct ctctaagttt agaagcgctc atctggcttttcgcttgctt ctgcagcaac 54cgact gttgtacaag ctcaggaggc gaataaatgt tcaaactgta tgctgatgtt 6atggga atttatttca aagaggaaaa gttaatattt tactttaaaa aaatcaaaat 66 665 DNA Homo sapiens tggatg ggtgctgttt agacaaacgc cgtctcctatataagacctg acagcacagg 6ctccg ccaggactgc aggcccacct gtctgcaacc cagctgaggc catgccctcc gggaccg tctgcagcct cctgctcctc ggcatgctct ggctggactt ggccatggca tccagct tcctgagccc tgaacaccag agagtccagg tgagacctcc ccacaaagcc 24tgttgttccagccct gccacttagc aaccagctct gtgacctgga gcagcagcgc 3tctggg cttcagtctt ctcccagagc acaaaggact ctgggtctga cctcactgtt 36aagga catgggggct tagagtccta aacagactgt ttcccccttc cagcagagaa 42tcgaa gaagccacca gccaagctgc agccccgagc tctagcaggctggctccgcc 48gatgg aggtcaagca gaaggggcag aggatgaact ggaagtccgg gtcggtacct 54gtttt atgcttctgt ggcagcgagg agggtgggg 579 DNA Homo sapiens tgcctg agactactcc agcctccccc gtccctgatg tcacaattca gaggctgctg 6ttagg aggttgtagaaagctctgta ggttctctct gtgtgtccta caggagtctt gccagct ccctgtcgga tggcttttat gaaaaaatat ctcctcccca ttctggggct catggcc tactactact attctgcaaa cgaggaattc agaccagaga tgctccaagg 24aagtg attgtcacag gggccagcaa agggatcgga agagagatgg cttatcatct3aagatg ggagcccatg tggtggtgac agcgaggtca aaagaaactc tacagaaggt 36cccac tgcctggagc ttggagcagc ctcagcacac tacattgctg gcaccatgga 42tgacc ttcgcagagc aatttgttgc ccaagcagga aagctcatgg gaggactaga 48tcatt ctcaaccaca tcaccaacacttctttgaat ctttttcatg atgatattca 54tgcgc aaaagcatgg aagtcaactt cctcagttac gtggtcctga ctgtagctgc 6cccatg ctgaagcaga gcaatggaag cattgttgtc gtctcctctc tggctgggaa 66cttat ccaatggttg ctgcctattc tgcaagcaag tttgctttgg atgggttctt 72ccatc agaaaggaat attcagtgtc cagggtcaat gtatcaatca ctctctgtgt 78gcctc atagacacag aaacagccat gaaggcagtt tctgggatag tccatatgca 84ctcca aaggaggaat gtgccctgga gatcatcaaa gggggagctc tgcgccaaga 9gtgtat tatgacagct cactctggac cactcttctgatcagaaatc catgcaggaa 96tggaa tttctctact caacgagcta taatatggac agattcataa acaagtagga tccctgag ggctgggcat gctgagggat tttgggactg ttctgtctca tgtttatctg ctcttatc tatgaagaca tcttcccaga gtgtccccag agacatgcaa gtcatgggtc acctgacaaatggaagga gttcctctaa catttgcaaa atggaaatgt aataataatg tgtcatgc accgctgcag ccagcagttg taaaattgtt agtaaacata ggtataatta agatagtt atattaaatt tatatcttat atataataat atgtgatgat taatacaata aattataa taaaggtcac ataaacttta taaattcataactggtagct ataacttgag tattcagg atggtttctt taaaaccata aactgtacaa atgaaatttt tcaatatttg tcttat 7Homo sapiens misc_feature (626)..(626) n is a, g, c, or t tgcctg agactactcc agcctccccc gtccctgatg tcacaattca gaggctgctg 6ttagg aggttgtaga aagctctgta ggttctctct gtgtgtccta caggagtctt gccagct ccctgtcgga tggcttttat gaaaaaatat ctcctcccca ttctggggct catggcc tactactact attctgcaaa cgaggaattc agaccagaga tgctccaagg 24aagtg attgtcacag gggccagcaa
agggatcgga agagagatgg cttatcatct 3aagatg ggagcccatg tggtggtgac agcgagctca gcacactaca ttgctggcac 36aagac atgaccttcg cagagcaatt tgttgcccaa gcaggaaagc tcatgggagg 42acatg ctcattctca accacatcac caacacttct ttgaatcttt ttcatgatga48accat gtgcgcaaaa gcatggaagt caacttcctc agttacgtgg tcctgactgt 54ccttg cccatgctga agcagagcaa tggaagcatg tgcgctcttc tgctggaatg 6catgtt gtgcatctga gcagtngttg atggtctctc tcatagaaga tatcaggcag 66atata ctttggtctg ctataccagacgctaggcgt ctgatgca 7394 DNA Homo sapiens tgcctg agactactcc agcctccccc gtccctgatg tcacaattca gaggctgctg 6ttagg aggttgtaga aagctctgta ggttctctct gtgtgtccta caggagtctt gccagct ccctgtcgga tggcttttat gaaaaaatat ctcctccccattctggggct catggcc tactactact attctgcaaa cgaggaattc agaccagaga tgctccaagg 24aagtg attgtcacag gggccagcaa agggatcgga agagagatgg cttatcatct 3aagatg ggagcccatg tggtggtgac agcgaggtca aaagaaactc tacagaaggt 36cccac tgcctggagcttggagcagc ctcagcacac tacattgctg gcaccatgga 42tgacc ttcgcagagc aatttgttgc ccaagcagga aagctcatgg gaggactaga 48tcatt ctcaaccaca tcaccaacac ttctttgaat ctttttcatg atgatattca 54tgcgc aaaagcatgg aagtcaactt cctcagttac gtggtcctga ctgtagctgc6cccatg ctgaagcaga gcaatggaag cattgttgtc gtctcctctc tggctgggaa 66cttat ccaatggttg ctgcctattc tgcaagcaag tttgctttgg atgggttctt 72ccatc agaaaggaat attcagtgtc cagggtcaat gtatcaatca ctctctgtgt 78gcctc atagacacag aaacagccatgaaggcagtt tctgggatag tccatatgca 84ctcca aaggaggaat gtgccctgga gatcatcaaa gggggagctc tgcgccaaga 9gtgtat tatgacagct cactctggac cactcttctg atcagaaatc catgcaggaa 96tggaa tttctctact caacgagcta taatatggag ggactgttct gtctcatgtt tctgagct cttatctatg aagacatctt cccagagtgt ccccagagac atgcaagtca ggtcacac ctgacaaatg gaaggagttc ctctaacatt tgcaaaatgg aaatgtaata aatgaatg tcatgcaccg ctgcagccag cagttgtaaa attgttagta aacataggta attaccag atagttatat taaatttatatcttatatat aataatatgt gatgattaat aatattaa ttataataaa ggtcacataa actttataaa ttcataactg gtagctataa tgagctta ttcaggatgg tttctttaaa accataaact gtacaaatga aatttttcaa tttgtttc ttat A Homo sapiens tgcctg agactactccagcctccccc gtccctgatg tcacaattca gaggctgctg 6ttagg aggttgtaga aagctctgta ggttctctct gtgtgtccta caggagtctt gccagct ccctgtcgga tggcttttat gaaaaaatat ctcctcccca ttctggggct catggcc tactactact attctgcaaa cgaggaattc agaccagaga tgctccaagg24aagtg attgtcacag gggccagcaa agggatcgga agagagatgg cttatcatct 3aagatg ggagcccatg tggtggtgac agcgaggtca aaagaaactc tacagaaggt 36cccac tgcctggagc ttggagcagc ctcagcacac tacattgctg gcaccatgga 42tgacc ttcgcagagc aatttgttgcccaagcagga aagctcatgg gaggactaga 48tcatt ctcaaccaca tcaccaacac ttctttgaat ctttttcatg atgatattca 54tgcgc cccatgctga agcagagcaa tggaagcatt gttgtcgtct cctctctggc 6aaagtg gcttatccaa tggttgctgc ctattctgca agcaagtttg ctttggatgg 66tctcc tccatcagaa aggaatattc agtgtccagg gtcaatgtat caatcactct 72ttctt ggcctcatag acacagaaac agccatgaag gcagtttctg ggatagtcca 78aagca gctccaaagg aggaatgtgc cctggagatc atcaaagggg gagctctgcg 84aagaa gtgtattatg acagctcact ctggaccactcttctgatca gaaatccatg 9aagatc ctggaatttc tctactcaac gagctataat atggacagat tcataaacaa 96aactc cctgagggct gggcatgctg agggattttg ggactgttct gtctcatgtt tctgagct cttatctatg aagacatctt cccagagtgt ccccagagac atgcaagtca ggtcacacctgacaaatg gaaggagttc ctctaacatt tgcaaaatgg aaatgtaata aatgaatg tcatgcaccg ctgcagccag cagttgtaaa attgttagta aacataggta attaccag atagttatat taaatttata tcttatatat aataatatgt gatgattaat aatattaa ttataataaa ggtcacataa actttataaattcataactg gtagctataa tgagctta ttcaggatgg tttctttaaa accataaact gtacaaatga aatttttcaa tttgtttc ttat A Homo sapiens tgcctg agactactcc agcctccccc gtccctgatg tcacaattca gaggctgctg 6ttagg aggttgtaga aagctctgtaggttctctct gtgtgtccta caggagtctt gccagct ccctgtcgga tggcttttat gaaaaaatat ctcctcccca ttctggggct catggcc tactactact attctgcaaa cgaggaattc agaccagaga tgctccaagg 24aagtg attgtcacag gggccagcaa agggatcgga agagagatgg cttatcatct 3aagatg ggagcccatg tggtggtgac agcgagctca gcacactaca ttgctggcac 36aagac atgaccttcg cagagcaatt tgttgcccaa gcaggaaagc tcatgggagg 42acatg ctcattctca accacatcac caacacttct ttgaatcttt ttcatgatga 48accat gtgcgcaaaa gcatggaagt caacttcctcagttacgtgg tcctgactgt 54ccttg cccatgctga agcagagcaa tggaagcatt gttgtcgtct cctctctggc 6aaagtg gcttatccaa tggttgctgc ctattctgca agcaagtttg ctttggatgg 66tctcc tccatcagaa aggaatattc agtgtccagg gtcaatgtat caatcactct 72ttcttggcctcatag acacagaaac agccatgaag gcagtttctg ggatagtcca 78aagca gctccaaagg aggaatgtgc cctggagatc atcaaagggg gagctctgcg 84aagaa gtgtattatg acagctcact ctggaccact cttctgatca gaaatccatg 9aagatc ctggaatttc tctactcaac gagctataat atggacagattcataaacaa 96aactc cctgagggct gggcatgctg agggattttg ggactgttct gtctcatgtt tctgagct cttatctatg aagacatctt cccagagtgt ccccagagac atgcaagtca ggtcacac ctgacaaatg gaaggagttc ctctaacatt tgcaaaatgg aaatgtaata aatgaatg tcatgcaccgctgcagccag cagttgtaaa attgttagta aacataggta attaccag atagttatat taaatttata tcttatatat aataatatgt gatgattaat aatattaa ttataataaa ggtcacataa actttataaa ttcataactg gtagctataa tgagctta ttcaggatgg tttctttaaa accataaact gtacaaatgaaatttttcaa tttgtttc ttat A Homo sapiens aaaagg gaaaacctgc ccaaatccag tttttgtttc agtaacttcc tttgagacaa 6ggaat ctgagagtaa gcacctgcta agggtgggac aggggctctg tctggtatgc tcccatg ttaagagcta acaatagtaa tggataagtctccagggcaa ccaggaccac caagcat tcctgtcttg ggctgcctcg agggctcctc tgtcctttgg ggagtactga 24gcctg atgcccagaa ctggcccact ctggcttctc tttggagctg tctctgcagg 3ttctgg ctgccagctc ggtcctagca taagggactt cttccttggc ctgggtttca 36ttgtatcaggtggca gaccagctgg tttcagtccc aaatcaggtc ttctgactcc 42gaaac caaccaactt ctgagcagga aatcctgccc ctccccaaag agtgggaaac 48aggaa gagagagatg aaacagaagg aaaggcagag gaggagggag agagagagaa 54gaaaa agaaaaaaga acatcaataa aaagaagtca gatttgttcgaaatcttgag 6gctcca aggaaagaaa gtgattgtca caggggccag caaagggatc ggaagagaga 66tatca tctggcgaag atgggagccc atgtggtggt gacagcgagg tcaaaagaaa 72cagaa ggtggtatcc cactgcctgg agcttggagc agcctcagca cactacattg 78accat ggaagacatgaccttcgcag agcaatttgt tgcccaagca ggaaagctca 84ggact agacatgctc attctcaacc acatcaccaa cacttctttg aatctttttc 9tgatat tcaccatgtg cgcaaaagca tggaagtcaa cttcctcagt tacgtggtcc 96gtagc tgccttgccc atgctgaagc agagcaatgg aagcattgtt gtcgtctcctctggctgg gaaagtggct tatccaatgg ttgctgccta ttctgcaagc aagtttgctt gatgggtt cttctcctcc atcagaaagg aatattcagt gtccagggtc aatgtatcaa actctctg tgttcttggc ctcatagaca cagaaacagc catgaaggca gtttctggga gtccatat gcaagcagct ccaaaggaggaatgtgccct ggagatcatc aaagggggag ctgcgcca agaagaagtg tattatgaca gctcactctg gaccactctt ctgatcagaa ccatgcag gaagatcctg gaatttctct actcaacgag ctataatatg gacagattca aacaagta ggaactccct gagggctggg catgctgagg gattttggga ctgttctgtc atgtttat ctgagctctt atctatgaag acatcttccc agagtgtccc cagagacatg agtcatgg gtcacacctg acaaatggaa ggagttcctc taacatttgc aaaatggaaa taataata atgaatgtca tgcaccgctg cagccagcag ttgtaaaatt gttagtaaac aggtataa ttaccagata gttatattaaatttatatct tatatataat aatatgtgat ttaataca atattaatta taataaaggt cacataaact ttataaattc ataactggta tataactt gagcttattc aggatggttt ctttaaaacc ataaactgta caaatgaaat ttcaatat ttgtttctta t A Homo sapiens tgcctgagactactcc agcctccccc gtccctgatg tcacaattca gaggctgctg 6ttagg aggttgtaga aagctctgta ggttctctct gtgtgtccta caggagtctt gccagct ccctgtcgga tggcttttat gaaaaaatat ctcctcccca ttctggggct catggcc tactactact attctgcaaa cgaggaattc agaccagagatgctccaagg 24aagtg attgtcacag gggccagcaa agggatcgga agagagatgg cttatcatct 3aagatg ggagcccatg tggtggtgac agcgaggtca aaagaaactc tacagaaggt 36cccac tgcctggagc ttggagcagc ctcagcacac tacattgctg gcaccatgga 42tgacc ttcgcagagcaatttgttgc ccaagcagga aagctcatgg gaggactaga 48tcatt ctcaaccaca tcaccaacac ttctttgaat ctttttcatg atgatattca 54tgcgc aaaagcatgg aagtcaactt cctcagttac gtggtcctga ctgtagctgc 6cccatg ctgaagcaga gcaatggaag cattgttgtc gtctcctctc tggctgaaac66tgaag gcagtttctg ggatagtcca tatgcaagca gctccaaagg aggaatgtgc 72agatc atcaaagggg gagctctgcg ccaagaagaa gtgtattatg acagctcact 78ccact cttctgatca gaaatccatg caggaagatc ctggaatttc tctactcaac 84ataat atggacagat tcataaacaagtaggaactc cctgagggct gggcatgctg 9attttg ggactgttct gtctcatgtt tatctgagct cttatctatg aagacatctt 96agtgt ccccagagac atgcaagtca tgggtcacac ctgacaaatg gaaggagttc ctaacatt tgcaaaatgg aaatgtaata ataatgaatg tcatgcaccg ctgcagccag gttgtaaa attgttagta aacataggta taattaccag atagttatat taaatttata ttatatat aataatatgt gatgattaat acaatattaa ttataataaa ggtcacataa tttataaa ttcataactg gtagctataa cttgagctta ttcaggatgg tttctttaaa cataaact gtacaaatga aatttttcaatatttgtttc ttat A Mus musculus ttggcc tctggawtca gaggctgctg cctgcctggg aggttgtaga aagctctgca 6tcttc gtgtgtccta cagggcgccc tgagccaggt ccctgtttga tggcagttat aaattac ctcctcccga tcctggtgct cttcctggcc tactactact attctacaaaagagttc agaccagaaa tgctccaggg aaagaaagtg attgtcactg gggccagcaa 24ttgga agagaaatgg catatcatct gtcaaaaatg ggagcccatg tggtattgac 3aggtcg gaggaaggtc tccagaaggt agtgtctcgc tgccttgaac tcggagcagc 36ctcac tacattgctg gcactatggaagacatgaca tttgcggagc aatttattgt 42cggga aagctcatgg gcggactgga catgcttatt ctaaaccaca tcactcagac 48tgtct ctcttccatg acgacatcca ctctgtgcga agagtcatgg aggtcaactt 54gctac gtggtcatga gcacagccgc cttgcccatg ctgaagcaga gcaatggcag 6gccgtc atctcctcct tggctgggaa aatgacccag cctatgattg ctccctactc 66gcaag tttgctctgg atgggttctt ttccaccatt agaacagaac tctacataac 72tcaac gtgtccatca ctctctgtgt ccttggcctc atagacacag aaacagctat 78aaatc tctgggataa ttaacgccca agcttctcccaaggaggagt gcgccctgga 84tcaaa ggcacagctc tacgcaaaag cgaggtgtac tatgacaaat cgcctttgac 9atcctg cttgggaacc caggaaggaa gatcatggaa tttttttcat tacgatatta 96aggac atgtttgtaa gtaactagga actcctgagc cctggtgagt ggtcttagaa gtcctgcctgatacttct gtaagcccta cccacaaaag tatctttcca gagatacaca ttttgggg tacacctcat catgagaaat tcttgcaaca cttgcacagt gaaaatgtaa gtaataaa tgtcacaaac cactttgggg cctgcagttg tgaacttgat tgtaactatg tataaaca catagtggtt gtatcggctt tacctcacactgaatgaaac aatgataact tgtaacat taaatataat aaaggtaata tcaactttgt aaatgca A Mus musculus 2tggcc tctggawtca gaggctgctg cctgcctggg aggttgtaga aagctctgca 6tcttc gtgtgtccta cagggcgccc tgagccaggt ccctgtttga tggcagttat aaattac ctcctcccga tcctggtgct cttcctggcc tactactact attctacaaa agagttc agactccaga aggtagtgtc tcgctgcctt gaactcggag cagcctctgc 24acatt gctggcacta tggaagacat gacatttgcg gagcaattta ttgtcaaggc 3aagctc atgggcggac tggacatgct tattctaaaccacatcactc agacctcgct 36tcttc catgacgaca tccactctgt gcgaagagtc atggaggtca acttcctcag 42tggtc atgagcacag ccgccttgcc catgctgaag cagagcaatg gcagcattgc 48tctcc tccttggctg ggaaaatgac ccagcctatg attgctccct actctgcaag 54ttgctctggatgggt tcttttccac cattagaaca gaactctaca taaccaaggt 6gtgtcc atcactctct gtgtccttgg cctcatagac acagaaacag ctatgaagga 66ctggg ataattaacg cccaagcttc tcccaaggag gagtgcgccc tggagatcat 72gcaca gctctacgca aaagcgaggt gtactatgac aaatcgcctttgactccaat 78ttggg aacccaggaa ggaagatcat ggaatttttt tcattacgat attataataa 84tgttt gtaagtaact aggaactcct gagccctggt gagtggtctt agaacagtcc 9tgatac ttctgtaagc cctacccaca aaagtatctt tccagagata cacaaatttt 96acacc tcatcatgagaaattcttgc aacacttgca cagtgaaaat gtaattgtaa aatgtcac aaaccacttt ggggcctgca gttgtgaact tgattgtaac tatggatata cacatagt ggttgtatcg gctttacctc acactgaatg aaacaatgat aactaatgta attaaata taataaaggt aatatcaact ttgtaaatgc a 845 DNAMus musculus 2tggcc tctggawtca gaggctgctg cctgcctggg aggttgtaga aagctctgca 6tcttc gtgtgtccta cagggcgccc tgagccaggt ccctgtttga tggcagttat aaattac ctcctcccga tcctggtgct cttcctggcc tactactact attctacaaa agagttc agaccagaaatgctccaggg aaagaaagtg attgtcactg gggccagcaa 24ttgga agagaaatgg catatcatct gtcaaaaatg ggagcccatg tggtattgac 3aggtcg gaggaaggtc tccagaaggt agtgtctcgc tgccttgaac tcggagcagc 36ctcac tacattgctg gcactatgga agacatgaca tttgcggagc aatttattgt42cggga aagctcatgg gcggactgga catgcttatt ctaaaccaca tcactcagac 48tgtct ctcttccatg acgacatcca ctctgtgcga agagtcatgg aggtcaactt 54gctac gtggtcatga gcacagccgc cttgcccatg ctgaagcaga gcaatggcag 6gccgtc atctcctcct tggctgggggaagaacagtt ccacaacaga gaagtcgcag 66ctcct gactcccgcg gcccgtgatt aatatcacca gccacagaat ggactggaac 72atcga tctggtggga ttggatataa cgaacataga attactcctg agactaccag 78aatag ttcaaatcaa atcatgccag aatatcagac aaatccaaat ggcaaaacag 84845 22 244 PRT Homo sapiens 22 Met Leu Leu Leu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro 2 Lys Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly 35 4sAsn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu 5 Lys Gly Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile 65 7 Gly Glu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly 85 9e Gln Gly Arg Lys Gly Glu Pro GlyGlu Gly Ala Tyr Val Tyr Arg Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp Ser Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr TyrPhe Ala Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu Val Gly 2Gln Val Trp Leu Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu 222la Asp Asn Asp Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr 225 234sp Thr Asn 23 Homo sapiens 23 Met Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly Ile Gln GlyArg Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg Ser Ala Phe Ser 2 Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met Pro Ile Arg Phe 35 4r Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp Gly Ser Thr Gly 5 Lys Phe His Cys AsnIle Pro Gly Leu Tyr Tyr Phe Ala Tyr His Ile 65 7 Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe Lys Lys Asp Lys 85 9a Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn Asn Val Asp Gln Ser Gly Ser Val Leu Leu His Leu Glu Val GlyAsp Gln Val Trp Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu Tyr Ala Asp Asn Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr His Asp Thr Asn 24 Homo sapiens 24 Met Leu Leu Leu Gly Ala Val Leu Leu Leu LeuAla Leu Pro Gly His Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro 2 Lys Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly 35 4s Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu 5 LysGly Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile 65 7 Gly Glu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly 85 9e Gln Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala Leu Leu Ser Pro Cys Pro Phe Ala Leu Pro ArgSer Ser Thr Ile Ser Lys Thr Thr Met Ala Pro Leu Val Asn Ser Thr Ala Thr Phe Leu Gly Cys Thr Leu Pro Thr Thr Ser Gln Ser Ile 25
Homo sapiens 25 Met Leu Leu Leu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro 2 Lys Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly 35 4s Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu 5 Lys Gly Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile 65 7 Gly Glu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly 85 9e Gln Gly Arg Lys Gly Glu ProGly Glu Gly Ala Tyr Val Tyr Arg Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp Ser Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu TyrLeu His Arg Leu Ser Ser Leu Pro 247 PRT Mus musculus 26 Met Leu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val 2 Pro Pro Pro Lys Gly Thr CysAla Gly Trp Met Ala Gly Ile Pro Gly 35 4s Pro Gly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr 5 Pro Gly Glu Lys Gly Glu Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys 65 7 Gly Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu Gly Pro ArgGly 85 9e Pro Gly Thr Pro Gly Arg Lys Gly Glu Pro Gly Glu Ala Ala Tyr Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn Tyr AspGly Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu Tyr Tyr Phe Ser Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Leu Phe Lys Lys Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr Glu Lys Asn Val Asp Gln AlaSer Gly Ser Val Leu Leu His Leu 2Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly Asp Gly Asp His 222ly Leu Tyr Ala Asp Asn Val Asn Asp Ser Thr Phe Thr Gly Phe 225 234eu Tyr His Asp Thr Asn 245 27 Musmusculus 27 Met Thr Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly Thr Pro Gly Arg Gly Glu Pro Gly Glu Ala Ala Tyr Val Tyr Arg Ser Ala Phe Ser 2 Val Gly Leu Glu Thr Arg Val Thr Val Pro Asn Val Pro Ile Arg Phe 35 4r Lys Ile Phe TyrAsn Gln Gln Asn His Tyr Asp Gly Ser Thr Gly 5 Lys Phe Tyr Cys Asn Ile Pro Gly Leu Tyr Tyr Phe Ser Tyr His Ile 65 7 Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe Lys Lys Asp Lys 85 9a Val Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Lys AsnVal Asp Gln Ser Gly Ser Val Leu Leu His Leu Glu Val Gly Asp Gln Val Trp Gln Val Tyr Gly Asp Gly Asp His Asn Gly Leu Tyr Ala Asp Asn Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr His Asp Thr Asn 28 Mus musculus 28 Met Leu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val 2 Pro Pro Pro Lys Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly 35 4sPro Gly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr 5 Pro Gly Glu Lys Gly Glu Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys 65 7 Gly Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly 85 9e Pro Gly Thr Pro Gly Arg Lys GlyGlu Pro Gly Glu Ala Ala Ser Phe Pro Met Tyr Pro Phe Ala Leu Leu Arg Ser Ser Thr Thr Asn Ile Ile Met Thr Ala Ala Leu Ala Ser Ser Thr Ala Thr Phe Arg Ser Thr Thr Ser Leu Thr Thr Ser Arg Cys Thr Mus musculus 29 Met Leu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val 2 Pro Pro Pro Lys Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly 35 4s ProGly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr 5 Pro Gly Glu Lys Gly Glu Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys 65 7 Gly Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly 85 9e Pro Gly Thr Pro Gly Arg Lys Gly GluPro Gly Glu Ala Ala Tyr Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn Tyr Asp Gly Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu Tyr Ile Tyr Trp Leu Ser Ser Leu Pro 76 PRT Mus musculus 3eu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val 2 Pro Pro Pro Lys Gly ThrCys Ala Gly Trp Met Ala Gly Ile Pro Gly 35 4s Pro Gly His Ile Lys Ile Lys Phe Glu Gly His Pro Pro Gly Arg 5 Leu Asn Cys Ala Lys Ile Trp His Phe Leu Gln Asp 65 7 Homo sapiens 3ro Ser Pro Gly Thr Val Cys Ser Leu Leu LeuLeu Gly Met Leu Leu Asp Leu Ala Met Ala Gly Ser Ser Phe Leu Ser Pro Glu His 2 Gln Arg Val Gln Gln Arg Lys Glu Ser Lys Lys Pro Pro Ala Lys Leu 35 4n Pro Arg Ala Leu Ala Gly Trp Leu Arg Pro Glu Asp Gly Gly Gln 5 Ala GluGly Ala Glu Asp Glu Leu Glu Val Arg Phe Asn Ala Pro Phe 65 7 Asp Val Gly Ile Lys Leu Ser Gly Val Gln Tyr Gln Gln His Ser Gln 85 9a Leu Gly Lys Phe Leu Gln Asp Ile Leu Trp Glu Glu Ala Lys Glu Pro Ala Asp Lys Homo sapiens 32 Met Pro Ser Pro Gly Thr Val Cys Ser Leu Leu Leu Leu Gly Met Leu Leu Asp Leu Ala Met Ala Gly Ser Ser Phe Leu Ser Pro Glu His 2 Gln Arg Val Gln Val Arg Pro Pro His Lys Ala Pro His Val Val Pro 35 4a Leu Pro LeuSer Asn Gln Leu Cys Asp Leu Glu Gln Gln Arg His 5 Trp Ala Ser Val Phe Ser Gln Ser Thr Lys Asp Ser Gly Ser Asp Leu 65 7 Thr Val Ser Gly Arg Thr Trp Gly Leu Arg Val Leu Asn Arg Leu Phe 85 9o Pro Ser Ser Arg Glu Arg Ser Arg Arg Ser HisGln Pro Ser Cys Pro Glu Leu 292 PRT Homo sapiens 33 Met Ala Phe Met Lys Lys Tyr Leu Leu Pro Ile Leu Gly Leu Phe Met Tyr Tyr Tyr Tyr Ser Ala Asn Glu Glu Phe Arg Pro Glu Met Leu 2 Gln Gly Lys Lys Val Ile Val ThrGly Ala Ser Lys Gly Ile Gly Arg 35 4u Met Ala Tyr His Leu Ala Lys Met Gly Ala His Val Val Val Thr 5 Ala Arg Ser Lys Glu Thr Leu Gln Lys Val Val Ser His Cys Leu Glu 65 7 Leu Gly Ala Ala Ser Ala His Tyr Ile Ala Gly Thr Met Glu Asp Met85 9r Phe Ala Glu Gln Phe Val Ala Gln Ala Gly Lys Leu Met Gly Gly Asp Met Leu Ile Leu Asn His Ile Thr Asn Thr Ser Leu Asn Leu His Asp Asp Ile His His Val Arg Lys Ser Met Glu Val Asn Phe Ser Tyr ValVal Leu Thr Val Ala Ala Leu Pro Met Leu Lys Gln Ser Asn Gly Ser Ile Val Val Val Ser Ser Leu Ala Gly Lys Val Ala Pro Met Val Ala Ala Tyr Ser Ala Ser Lys Phe Ala Leu Asp Gly Phe Ser Ser Ile Arg Lys Glu TyrSer Val Ser Arg Val Asn Val 2Ile Thr Leu Cys Val Leu Gly Leu Ile Asp Thr Glu Thr Ala Met 222la Val Ser Gly Ile Val His Met Gln Ala Ala Pro Lys Glu Glu 225 234la Leu Glu Ile Ile Lys Gly Gly Ala Leu Arg Gln GluGlu Val 245 25yr Tyr Asp Ser Ser Leu Trp Thr Thr Leu Leu Ile Arg Asn Pro Cys 267ys Ile Leu Glu Phe Leu Tyr Ser Thr Ser Tyr Asn Met Asp Arg 275 28he Ile Asn Lys 293 PRT Homo sapiens MISC_FEATURE (a can beany naturally occuring amino acid 34 Met Ala Phe Met Lys Lys Tyr Leu Leu Pro Ile Leu Gly Leu Phe Met Tyr Tyr Tyr Tyr Ser Ala Asn Glu Glu Phe Arg Pro Glu Met Leu 2 Gln Gly Lys Lys Val Ile Val Thr Gly Ala Ser Lys Gly Ile Gly Arg 35 4u Met Ala Tyr His Leu Ala Lys Met Gly Ala His Val Val Val Thr 5 Ala Ser Ser Ala His Tyr Ile Ala Gly Thr Met Glu Asp Met Thr Phe 65 7 Ala Glu Gln Phe Val Ala Gln Ala Gly Lys Leu Met Gly Gly Leu Asp 85 9t Leu Ile Leu Asn His IleThr Asn Thr Ser Leu Asn Leu Phe His Asp Ile His His Val Arg Lys Ser Met Glu Val Asn Phe Leu Ser Val Val Leu Thr Val Ala Ala Leu Pro Met Leu Lys Gln Ser Asn Ser Met Cys Ala Leu Leu Leu Glu Cys Tyr His ValVal His Leu Ser Ser Xaa 35 295 PRT Homo sapiens 35 Met Ala Phe Met Lys Lys Tyr Leu Leu Pro Ile Leu Gly Leu Phe Met Tyr Tyr Tyr Tyr Ser Ala Asn Glu Glu Phe Arg Pro Glu Met Leu 2 Gln Gly Lys Lys Val Ile Val Thr Gly AlaSer Lys Gly Ile Gly Arg 35 4u Met Ala Tyr His Leu Ala Lys Met Gly Ala His Val Val Val Thr 5 Ala Arg Ser Lys Glu Thr Leu Gln Lys Val Val Ser His Cys Leu Glu 65 7 Leu Gly Ala Ala Ser Ala His Tyr Ile Ala Gly Thr Met Glu Asp Met 85 9r Phe Ala Glu Gln Phe Val Ala Gln Ala Gly Lys Leu Met Gly Gly Asp Met Leu Ile Leu Asn His Ile Thr Asn Thr Ser Leu Asn Leu His Asp Asp Ile His His Val Arg Lys Ser Met Glu Val Asn Phe Ser Tyr Val Val LeuThr Val Ala Ala Leu Pro Met Leu Lys Gln Ser Asn Gly Ser Ile Val Val Val Ser Ser Leu Ala Gly Lys Val Ala Pro Met Val Ala Ala Tyr Ser Ala Ser Lys Phe Ala Leu Asp Gly Phe Ser Ser Ile Arg Lys Glu Tyr Ser ValSer Arg Val Asn Val 2Ile Thr Leu Cys Val Leu Gly Leu Ile Asp Thr Glu Thr Ala Met 222la Val Ser Gly Ile Val His Met Gln Ala Ala Pro Lys Glu Glu 225 234la Leu Glu Ile Ile Lys Gly Gly Ala Leu Arg Gln Glu Glu Val245 25yr Tyr Asp Ser Ser Leu Trp Thr Thr Leu Leu Ile Arg Asn Pro Cys 267ys Ile Leu Glu Phe Leu Tyr Ser Thr Ser Tyr Asn Met Glu Gly 275 28eu Phe Cys Leu Met Phe Ile 296 274 PRT Homo sapiens 36 Met Ala Phe Met Lys Lys TyrLeu Leu Pro Ile Leu Gly Leu Phe Met Tyr Tyr Tyr Tyr Ser Ala Asn Glu Glu Phe Arg Pro Glu Met Leu 2 Gln Gly Lys Lys Val Ile Val Thr Gly Ala Ser Lys Gly Ile Gly Arg 35 4u Met Ala Tyr His Leu Ala Lys Met Gly Ala His Val Val ValThr 5 Ala Arg Ser Lys Glu Thr Leu Gln Lys Val Val Ser His Cys Leu Glu 65 7 Leu Gly Ala Ala Ser Ala His Tyr Ile Ala Gly Thr Met Glu Asp Met 85 9r Phe Ala Glu Gln Phe Val Ala Gln Ala Gly Lys Leu Met Gly Gly Asp Met LeuIle Leu Asn His Ile Thr Asn Thr Ser Leu Asn Leu His Asp Asp Ile His His Val Arg Pro Met Leu Lys Gln Ser Asn Ser Ile Val Val Val Ser Ser Leu Ala Gly Lys Val Ala Tyr Pro Met Val Ala Ala Tyr Ser Ala Ser LysPhe Ala Leu Asp Gly Phe Phe Ser Ile Arg Lys Glu Tyr Ser Val Ser Arg Val Asn Val Ser Ile Leu Cys Val Leu Gly Leu Ile Asp Thr Glu Thr Ala Met Lys Ala 2Ser Gly Ile Val His Met Gln Ala Ala Pro Lys Glu Glu CysAla 222lu Ile Ile Lys Gly Gly Ala Leu Arg Gln Glu Glu Val Tyr Tyr 225 234er Ser Leu Trp Thr Thr Leu Leu Ile Arg Asn Pro Cys Arg Lys 245 25le Leu Glu Phe Leu Tyr Ser Thr Ser Tyr Asn Met Asp Arg Phe Ile 267ys 37 274 PRT Homo sapiens 37 Met Ala Phe Met Lys Lys Tyr Leu Leu Pro Ile Leu Gly Leu Phe Met Tyr Tyr Tyr Tyr Ser Ala Asn Glu Glu Phe Arg Pro Glu Met Leu 2 Gln Gly Lys Lys Val Ile Val Thr Gly Ala Ser Lys Gly Ile Gly Arg 35 4uMet Ala Tyr His Leu Ala Lys Met Gly Ala His Val Val Val Thr 5 Ala Ser Ser Ala His Tyr Ile Ala Gly Thr Met Glu Asp Met Thr Phe 65 7 Ala Glu Gln Phe Val Ala Gln Ala Gly Lys Leu Met Gly Gly Leu Asp 85 9t Leu Ile Leu Asn His Ile Thr AsnThr Ser Leu Asn Leu Phe His Asp Ile His His Val Arg Lys Ser Met Glu Val Asn Phe Leu Ser Val Val Leu Thr Val Ala Ala Leu Pro Met Leu Lys Gln Ser Asn Ser Ile Val Val Val Ser Ser Leu Ala Gly Lys Val Ala TyrPro Met Val Ala Ala Tyr Ser Ala Ser Lys Phe Ala Leu Asp Gly Phe Phe
Ser Ile Arg Lys Glu Tyr Ser Val Ser Arg Val Asn Val Ser Ile Leu Cys Val Leu Gly Leu Ile Asp Thr Glu Thr Ala Met Lys Ala 2Ser Gly Ile Val His Met Gln Ala Ala Pro Lys Glu Glu Cys Ala 222lu Ile Ile Lys Gly Gly Ala Leu Arg Gln Glu Glu Val Tyr Tyr 225 234er Ser Leu Trp Thr Thr Leu Leu Ile Arg Asn Pro Cys Arg Lys 245 25le Leu Glu Phe Leu Tyr Ser Thr Ser Tyr Asn Met Asp Arg Phe Ile 267ys 38 262 PRT Homosapiens 38 Met Leu Gln Gly Lys Lys Val Ile Val Thr Gly Ala Ser Lys Gly Ile Arg Glu Met Ala Tyr His Leu Ala Lys Met Gly Ala His Val Val 2 Val Thr Ala Arg Ser Lys Glu Thr Leu Gln Lys Val Val Ser His Cys 35 4u Glu Leu Gly Ala AlaSer Ala His Tyr Ile Ala Gly Thr Met Glu 5 Asp Met Thr Phe Ala Glu Gln Phe Val Ala Gln Ala Gly Lys Leu Met 65 7 Gly Gly Leu Asp Met Leu Ile Leu Asn His Ile Thr Asn Thr Ser Leu 85 9n Leu Phe His Asp Asp Ile His His Val Arg Lys Ser MetGlu Val Phe Leu Ser Tyr Val Val Leu Thr Val Ala Ala Leu Pro Met Leu Gln Ser Asn Gly Ser Ile Val Val Val Ser Ser Leu Ala Gly Lys Ala Tyr Pro Met Val Ala Ala Tyr Ser Ala Ser Lys Phe Ala Leu Asp Gly Phe Phe Ser Ser Ile Arg Lys Glu Tyr Ser Val Ser Arg Val Val Ser Ile Thr Leu Cys Val Leu Gly Leu Ile Asp Thr Glu Thr Met Lys Ala Val Ser Gly Ile Val His Met Gln Ala Ala Pro Lys 2Glu Cys Ala Leu GluIle Ile Lys Gly Gly Ala Leu Arg Gln Glu 222al Tyr Tyr Asp Ser Ser Leu Trp Thr Thr Leu Leu Ile Arg Asn 225 234ys Arg Lys Ile Leu Glu Phe Leu Tyr Ser Thr Ser Tyr Asn Met 245 25sp Arg Phe Ile Asn Lys 264 PRT Homosapiens 39 Met Ala Phe Met Lys Lys Tyr Leu Leu Pro Ile Leu Gly Leu Phe Met Tyr Tyr Tyr Tyr Ser Ala Asn Glu Glu Phe Arg Pro Glu Met Leu 2 Gln Gly Lys Lys Val Ile Val Thr Gly Ala Ser Lys Gly Ile Gly Arg 35 4u Met Ala Tyr His LeuAla Lys Met Gly Ala His Val Val Val Thr 5 Ala Arg Ser Lys Glu Thr Leu Gln Lys Val Val Ser His Cys Leu Glu 65 7 Leu Gly Ala Ala Ser Ala His Tyr Ile Ala Gly Thr Met Glu Asp Met 85 9r Phe Ala Glu Gln Phe Val Ala Gln Ala Gly Lys Leu MetGly Gly Asp Met Leu Ile Leu Asn His Ile Thr Asn Thr Ser Leu Asn Leu His Asp Asp Ile His His Val Arg Lys Ser Met Glu Val Asn Phe Ser Tyr Val Val Leu Thr Val Ala Ala Leu Pro Met Leu Lys Gln Ser Asn Gly Ser Ile Val Val Val Ser Ser Leu Ala Glu Thr Ala Met Ala Val Ser Gly Ile Val His Met Gln Ala Ala Pro Lys Glu Glu Ala Leu Glu Ile Ile Lys Gly Gly Ala Leu Arg Gln Glu Glu Val 2Tyr Asp Ser Ser LeuTrp Thr Thr Leu Leu Ile Arg Asn Pro Cys 222ys Ile Leu Glu Phe Leu Tyr Ser Thr Ser Tyr Asn Met Asp Arg 225 234le Asn Lys 4RT Mus musculus 4la Val Met Lys Asn Tyr Leu Leu Pro Ile Leu Val Leu Phe Leu Tyr Tyr Tyr Tyr Ser Thr Asn Glu Glu Phe Arg Pro Glu Met Leu 2 Gln Gly Lys Lys Val Ile Val Thr Gly Ala Ser Lys Gly Ile Gly Arg 35 4u Met Ala Tyr His Leu Ser Lys Met Gly Ala His Val Val Leu Thr 5 Ala Arg Ser Glu Glu Gly Leu Gln Lys ValVal Ser Arg Cys Leu Glu 65 7 Leu Gly Ala Ala Ser Ala His Tyr Ile Ala Gly Thr Met Glu Asp Met 85 9r Phe Ala Glu Gln Phe Ile Val Lys Ala Gly Lys Leu Met Gly Gly Asp Met Leu Ile Leu Asn His Ile Thr Gln Thr Ser Leu Ser Leu His Asp Asp Ile His Ser Val Arg Arg Val Met Glu Val Asn Phe Ser Tyr Val Val Met Ser Thr Ala Ala Leu Pro Met Leu Lys Gln Ser Asn Gly Ser Ile Ala Val Ile Ser Ser Leu Ala Gly Lys Met Thr Pro MetIle Ala Pro Tyr Ser Ala Ser Lys Phe Ala Leu Asp Gly Phe Ser Thr Ile Arg Thr Glu Leu Tyr Ile Thr Lys Val Asn Val 2Ile Thr Leu Cys Val Leu Gly Leu Ile Asp Thr Glu Thr Ala Met 222lu Ile Ser Gly Ile Ile Asn AlaGln Ala Ser Pro Lys Glu Glu 225 234la Leu Glu Ile Ile Lys Gly Thr Ala Leu Arg Lys Ser Glu Val 245 25yr Tyr Asp Lys Ser Pro Leu Thr Pro Ile Leu Leu Gly Asn Pro Gly 267ys Ile Met Glu Phe Phe Ser Leu Arg Tyr Tyr Asn LysAsp Met 275 28he Val Ser Asn 29us musculus 4la Val Met Lys Asn Tyr Leu Leu Pro Ile Leu Val Leu Phe Leu Tyr Tyr Tyr Tyr Ser Thr Asn Glu Glu Phe Arg Leu Gln Lys Val 2 Val Ser Arg Cys Leu Glu Leu Gly Ala AlaSer Ala His Tyr Ile Ala 35 4y Thr Met Glu Asp Met Thr Phe Ala Glu Gln Phe Ile Val Lys Ala 5 Gly Lys Leu Met Gly Gly Leu Asp Met Leu Ile Leu Asn His Ile Thr 65 7 Gln Thr Ser Leu Ser Leu Phe His Asp Asp Ile His Ser Val Arg Arg 85 9l Met Glu Val Asn Phe Leu Ser Tyr Val Val Met Ser Thr Ala Ala Pro Met Leu Lys Gln Ser Asn Gly Ser Ile Ala Val Ile Ser Ser Ala Gly Lys Met Thr Gln Pro Met Ile Ala Pro Tyr Ser Ala Ser Phe Ala Leu Asp GlyPhe Phe Ser Thr Ile Arg Thr Glu Leu Tyr Ile Thr Lys Val Asn Val Ser Ile Thr Leu Cys Val Leu Gly Leu Ile Thr Glu Thr Ala Met Lys Glu Ile Ser Gly Ile Ile Asn Ala Gln Ser Pro Lys Glu Glu Cys Ala Leu Glu IleIle Lys Gly Thr Ala 2Arg Lys Ser Glu Val Tyr Tyr Asp Lys Ser Pro Leu Thr Pro Ile 222eu Gly Asn Pro Gly Arg Lys Ile Met Glu Phe Phe Ser Leu Arg 225 234yr Asn Lys Asp Met Phe Val Ser Asn 245 252 PRT Musmusculus 42 Met Ala Val Met Lys Asn Tyr Leu Leu Pro Ile Leu Val Leu Phe Leu Tyr Tyr Tyr Tyr Ser Thr Asn Glu Glu Phe Arg Pro Glu Met Leu 2 Gln Gly Lys Lys Val Ile Val Thr Gly Ala Ser Lys Gly Ile Gly Arg 35 4u Met Ala Tyr HisLeu Ser Lys Met Gly Ala His Val Val Leu Thr 5 Ala Arg Ser Glu Glu Gly Leu Gln Lys Val Val Ser Arg Cys Leu Glu 65 7 Leu Gly Ala Ala Ser Ala His Tyr Ile Ala Gly Thr Met Glu Asp Met 85 9r Phe Ala Glu Gln Phe Ile Val Lys Ala Gly Lys LeuMet Gly Gly Asp Met Leu Ile Leu Asn His Ile Thr Gln Thr Ser Leu Ser Leu His Asp Asp Ile His Ser Val Arg Arg Val Met Glu Val Asn Phe Ser Tyr Val Val Met Ser Thr Ala Ala Leu Pro Met Leu Lys Gln Ser Asn Gly Ser Ile Ala Val Ile Ser Ser Leu Ala Gly Gly Arg Thr Pro Gln Gln Arg Ser Arg Ser Val Thr Pro Asp Ser Arg Gly Pro >
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